Test of a model of decision processes in an intense conflict situation.

A particularly intense form of conflict. It is a situation in which at most one of some set of participants may obtain an objective and in which it is possible that none will obtain their objective. A mathematical model is derived from basic assumptions about the decision making process in a three-person uelative conflict situation, a truel. It is proposed that the model is generalizable to n-person situations. A preliminary test of the model that varied only one of the parameters revealed support for the model. More extensive tests of the model are warranted. In addition, the applicability of the model to internation or intergroup conflict is explored.


The absorption and metabolism in rats of small oral doses of dimethylnitrosamine. Implication for the possible hazard of dimethylnitrosamine in human food.

1. Groups of rats were given one dose of the carcinogen dimethylnitrosamine by gastric intubation. The dose was varied between 10mg/kg body wt. and 1 microgram/kg body wt. 2. The dose was rapidly absorbed. 3. The methylation of liver DNA resulting from the administration of this carcinogen was proportional to dose. This suggests that small doses are absorbed from the gut with no more loss than large doses. 4. As the dose was decreased there was a disproportionately greater decrease in the alkylation of kidney DNA, and when the dose was less than 40 microgram/kg body wt. the methylation of kidney DNA was no longer detectable. This possibly explains why small amounts of dimethylnitrosamine in the diet do not induce kidney tumours. 5. Comparison of the relative alkylation of liver DNA and kidney DNA resulting from an oral and from an intravenous dose of dimethylnitrosamine suggest that small amounts of dimethylnitrosamine absorbed into the portal blood from the gut are completely metabolized by the liver and do not enter the general circulation. 6. The implications of these results for the possible hazard of dimethylnitrosamine in human food is discussed.


A new method for assaying rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and its application in a study of the effect of dietary cholesterol on this effect of dietary cholesterol on this enzyme.

A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.


Effects of lactation of ketogenesis from oleate or butyrate in rat hepatocytes.

1. Rates of ketogenesis from endogenous butyrate or oleate were measured in isolated hepatocytes prepared from fed rats during different reproductive states [virgin, pregnant, early-lactating (2-4 days) and peak-lactating (10-17 days)]. In the peak-lactation group there was a decrease (25%) in the rate of ketogenesis from butyrate, but there were no differences in the rates between the other groups. Wth oleate, the rate of ketogenesis was increased in the pregnant and in the early-lactation groups compared with the virgin group, whereas the rate was 50% lower in the peak-lactation group. 2. Experiments with [1-(14)C]oleate indicated that these differences in rates of ketogenesis were not due to alterations in the rate of oleate utilization, but to changes in the amount of oleoyl-CoA converted into ketone bodies. 3. Although the addition of carnitine increased the rates of ketogenesis from oleate in all groups of rats, it did not abolish the differences between the groups. 4. Measurements of the accumulation of glucose and lactate showed that hepatocytes from rats at peak lactation had a higher rate of glycolytic flux than did hepatocytes from the other groups. After starvation, the rate of ketogenesis from oleate was still lower in the peak-lactation group compared with the control group. This suggests that the alteration in ketogenic capacity in the former group is not merely due to a higher glycolytic flux. 5. It is concluded that livers from rats at peak lactation have a lower capacity to produce ketone bodies from long-chain fatty acids which is due to an alteration in the partitioning of long-chain acyl-CoA esters between the pathways of triacylglycerol synthesis and beta-oxidation. The physiological relevance of this finding is discussed.


Hypoketonaemic effect of L-alamine. Specific decrease in blood concentrations of 3-hydroxybutyrate in the rat.

1. The injection of L-alanine (50-100 mg/kg) into 35-day-old rats that had been starveed for 48 h increased blood L-alanine concentration to values observed in fed animals and lowered the blood concentration of 3-hydroxybutyrate within 2 min. 2. This hypoketon aemic action of L-alanine was specific for 3-hydroxybutyrate, since the acetoacetate concentrations did not change significantly. 3. The decrease in 3-hydroxybutyrate elicited by L-alanine was not related to changes in the blood concentrations of insulin, glucagon, growth hormone, glucose, unesterified fatty acids, lactate or pyruvate. 4. The injection of L-alanine resulted in a decrease in total ketones that was apparently unrelated to their increased peripheral utilization. These results are interpreted as an anti-ketogenic action of L-alanine. 5. The data suggest that L-alamine lowers ketone-body formation in starved rats, possibly via an alteration in hepatic redox equilibrium.


Changes in surface-membrane components during the differentation of rabbit erythroid cells.

The membrane components of rabbit bone-marrow-bound erythroid cells were characterized and compared with those of circulating rabbit erythroid cells. By the criteria of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, radioiodination with lactoperoxidase and binding of radioiodinated lectins, the two circulating forms of erythroid cells (the reticulocyte and erythrocyte) have the same surface components. In contrast, bone-marrow-bound nucleated erythroid cells have a unique set of membrane surface components which are completely different from those found on circulating cells. Of the ten Coomassie-Blue-staining proteins present in nucleated erythroid-cell plasma-membrane preparations, eight are accessible at the extracellular surface, and all of these are lectin-binding glycoproteins. Bone-marrow erythroid cells separated according to age by velocity sedimentation were also studied. The changeover in surface components occurs after the last nucleated stage of the erythroid cells (the orthochromatic normoblast). We discuss the alterations in membrane surface components observed during the differentiation of the erythroid-cell series in relation to the transition from bone-marrow-bound to circulating forms of these cells. We suggest that the change in membrane surface components may be linked to the loss of the nucleus from the normoblast and the entry of the erythroid cell into the circulation.


Biosynthesis of the pyrimidinyl amino acid lathyrine by Lathyrus tingitanus L.

The biosynthesis of the pyrimidinyl amino acid lathyrine by seedlings of Lathyrus tingitanus L. was shown to be stimulated by uracil. [6(-14)C]Orotate, [2(-14)C]uracil and [3(-14)C]serine were incorporated into lathyrine; the incorporation of [6(-14)C]orotate was substantially decreased in the presence of uracil. Chemical degradation to locate the 14C incorporated from labelled precursors showed that 90% of the radioactivity incorporated into lathyrine from [3(-14)C]serine could be recovered in the alanine side chain. Over 80% of the radioactivity incorporated from [2(-14)C]uracil was shown to be located in C-2 of lathyrine. It is concluded that under the conditions studied, lathyrine arises from a preformed pyrimidine arising via the orotate pathway. Paradoxically, it was also possible to confirm previous reports that radioactivity from L-[guanidino-14C]homoarginine is incorporated into lathyrine and gamma-hydroxyhomoarginine. However, as homoarginine and gamma-hydroxyhomoarginine are also both labelled by [2(-14)C]uracil, it is suggested that they are products of the ring-opening of lathyrine and that reversibility of this process accounts, at least in part, for their observed experimental incorporation into lathyrine.


The metabolism of cyclohexanecarboxylate in the rat.

In the rat, cyclohexanecarboxylate was metabolized and excreted (mostly in the urine) as hippurate, hexahydrohippurate, 3,4,5,6-tetrahydrohippurate and benzoyl and cyclohexylcarbonyl beta-glucuronides. The pattern of metabolism is dose-dependent. With decreasing dose a progressive increase in the conversion into hippurate occurred. This was largely at the expense of glucuronide formation, although the proportions of hexahydro- and tetrahydro-hippurate were also decreased. The observed formation of hexahydrohippurate and 3,4,5,5-tetrahydrohippurate substantiates the proposed mechanism of aromatization of cyclohexanecarboxylate.  It appears that these compounds arise via glycine conjugation of active intermediates in the aromatization process. Hexahydrohippurate and 3,4,5,6-tetrahydrohippurate may occur in the urine of rats as new mitabolities of shikimate, dependent for their formation on microbial metabolism.


Retained folates in the rat.

The retention of radioactivity after doses of 14C- and 3H-labelled folic acid is described. Radioactivity was retained in liver, kidney and gut of rats for some time after administration of the dose. The retained radioactivity could not be displaced by large doses of unlabelled folic acid or unlabelled 5-methyltetrahydrofolate. 14C- and 3H-labbelled folates showed similar chromatographic behaviour onion-exchange chromatography to 5-methyltetrahydrofolate, and on ion-exchange and gel-permeation chromatography to synthetic pteroylhepta-gamma-glutamate.


A spectrophotometric procedure for rapid and sensitive measurements of beta-oxidation. Demonstration of factors that can be rate-limiting for beta-oxidation.

1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC 1.3.99.3). The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.


Interaction of the anti-oestrogen, nafoxidine hydrochloride, with the soluble nuclear oestradiol-binding protein in chick liver.

Nafoxidine hydrochloride (Upjohn, 11100A)injected with oestradiol into immature chicks inhibits the hormone-induced increase in [3H]oestradiol-binding activity in salt extracts of liver nuclei as well as the subsequent production by liver of egg-yolk phosphoprotein. Substantial inhibition of both oestradiol-induced responses is seen when nafoxidine is given in a dose approximately equimolar with that of oestradiol. In vitro nafoxidine competitively inhibits binding of [3H]oestradiol in nuclear extracts. The Ki for the inhibition is 43 nM, which indicates an affinity of nafoxidine for the binding protein about 4% of that of oestradiol. The inhibitory action of nafoxidine in vivo thus is more potent than the relative binding affinity determined in vitro might indicate. One possible explanation is that the primary site of nafoxidine action is at a point proximal to nuclear receptor interaction. Nafoxidine injected alone into the chick does not induce phosphoprotein synthesis, but it does increase [3H]oestradiol-binding activity in extracts of liver nuclei to a limited extent. No differences in the properties of the oestradiol-binding activity in extracts from nafoxidine-treated chicks or from oestradiol-treated chicks were detected. Chick liver cytosol does not contain detectable high-affinity oestradiol-binding activity. A low-affinity oestradiol-binding component with a sedimentation coefficient of 3.5S was found, but it was unaffected by treatment of chicks with earlier nafoxidine or oestradiol. The results suggest a difference in the mechanism of oestradiol action in the chick liver and in the widely studied rat uterus, on which the usual model for oestradiol action is largely based.


Number and activity of active ribosomes in bacterial polyribosomes.

Polyribosomes were isolated from Baccillus licheniformis, grown at 37 and 46 degrees C, and from Bacillus stearothermophilus, grown at 46 and 55 degrees C. The polyribosomes were incubated with either [3H]puromycin or [14C]phenylalanine. The number of active ribosomes (i.e. those to which growing polypeptide chains are attached) was calculated from the amount of [3H]peptidyl-puromycin formed. The activity of an active ribosome (i.e. the total number of amino acid molecules incorporated/unit time per active ribosome) was calculated from the uptake of [14C]phenylalanine. The number of active ribosomes per migrogram of RNA was as follows: for B. licheniformis, 1.66 X 10(12) and 1.72 X 10(12) at 37 and 46 degrees C respectively; for B. stearothermophilus, 2.59 X 10(12) at 46 and 55 degreesC respectively. The activity per active ribosome was as follows: for B. licheniformis, 0.61 and 0.05 at 37 and 46 degrees C respectively; for B. stearothermophilus, 0.58 and 0.42 at 46 and 55 degrees C respectively.


Selective permeability of rat liver mitochondria to purified aspartate aminotransferases in vitro.

1. A method was devised to allow determination of intramitochondrial aspartate amino-transferase activity in suspensions of intact mitochondria. 2. Addition of purified rat liver mitochondrial aspartate aminotransferase to suspensions of rat liver mitochondria caused an apparent increase in the intramitochondrial enzyme activity. No increase was observed when the mitochondria were preincubated with the purified cytoplasmic isoenzyme. 3. These results suggest that mitochondrial aspartate aminotransferase, but not the cytoplasmic isoenzyme, is able to pass from solution into the matrix of intact rat liver mitochondria in vitro. 4. This system may provide a model for studies of the little-understood processes by which cytoplasmically synthesized components are incorporated into mitochondria in vivo.


Pyruvate dehydrogenase activity in hamster small intestine during development.

Total pyruvate dehydrogenase activities in hamster intestine increase from 40 nmol/min (munits) per g of intestine in the foetal animals to 460 munits/g in the adult, whereas the fraction of the enzyme in the active form increases from 34 to 42% of the total activity over the same period. However, a complete conversion of the enzyme into the active form is observed in the neonatal animal immediately after birth. Results from experiments in vitro suggested that the activation of pyruvate dehydrogenase is controlled, in part, by the [NAD+]/[NADH] ratio. This proposal was tested in vivo by examining the proportion of the enzyme in the active form during conditions when the [NAD+]/[NADH] ratio was markedly altered, and the data show a direct relationship between the mitochondrial redox state and activity of the active form.


Adaptation of protein metabolism to endurance training. Increased amino acid oxidation in response to training.

This study was conducted to investigate alterations in excretion of urea and total nitrogen after6-8 weeks of daily exercise and to establish if the capacity for amino acid oxidation in muscle is influenced by endurance training. Urea nitrogen excretion was increased in trained compared with untrained rats and nitrogen balance was less positive in trained than in untrained rats. Increased [14C]leucine oxidation with training was observed both in vivo and in vitro. The results of this study demonstrate that amino acid catabolism is increased during exercise training and that the muscle enzymes involved in leucine oxidation adapt to endurance training in a manner similar to the enzymes of carbohydrate and fat catabolism.


Microbiological degradation of bile acids. The preparation of hexahydroindane derivatives as substrates for studying cholic acid degradation.

Relatively large amounts of 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb), which is believed to be one of the intermediates involved in the degradation of cholic acid (I), were needed to identify is further degradation products. A simple method for the preparation of this compound was then investigated. Arthrobacter simplex could degrade-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIa) to 3-(1beta-carboxy-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVa) in good yield, the structure of which was established by partial synthesis. It was therefore expected that, if a similar degradation by this organism occurred with 17alpha-hydroxy-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIb), which is easily obtained by chemical oxidation of commercially availabe 17alpha-hydroxydeoxycorticosterone, the resulting product, 3-(1beta-carboxy-3aalpha-hexahydro-1alpha-hydroxy-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVb), could be readily converted chemically into the required dioxocarboxylic acid, (IIb). Exposure of compound (IIIb) to A. simplex produced, as expected, compound (IVb) which was then oxidized with NaBiO3 to give a reasonable yield of compound (IIb).


Microbiological degradation of bile acids. Metabolites formed from 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl) propionic acid by Streptomyces rubescens.

1. The metabolism of 3-(3a alpha-hexahydro-7a beta-methyl-1,5-dioxoindan-4 alpha-yl)propionic acid (III), which is a possible precursor of 2,3,4,6,6a beta, 7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-1H-cyclopenta[f]quinoline-3,7-dione (II) formed from cholic acid (I) by streptomyces rubescens, was investigated by using the same organism. 2. This organism effected amide bond formation, reduction of the carbonyl groups, trans alpha beta-desaturation and R-oriented beta-hydroxylation of the propionic acid side chain and skeleton cleavage, and the following metabolites were isolated as these forms or their derivatives: compound (II), 1,2,3,4 a beta,-5,6,6a beta,7,8,9a alpha,9b beta-dodecahydro-6a beta -methylcyclopental[f][1]benzopyran-3,7-dione (IVa), (1R)-1,2,3,4a beta,5,6,6a beta,7,8,9.9a alpha,9b beta-dodecahydro-1-hydroxy-6a beta-methylcyclopenta[f][1]benzopyran-3,7-dione (IVb), (E)-3-(3aalpha-hexahydro-5 alpha-hydroxy-7a beta-methyl-l-oxo-indan-4 alpha-yl)prop-2-enoic acid (V), (+)-(5R)-5-methyl-4-oxo-octane-1,8-dioic acid (VI), 3-(4-hydroxy-5-methyl-2-oxo-2H-pyran-6-yl)propionic acid (VII) and 3-(3a alpha-hexahydro-1 beta-hydroxy-7a beta-methyl-5-oxoindan-4 alpha-yl)propionic acid (VIII). The metabolites (IVb), (V), (VI) and (VII) were new compounds, and their structures were established by chemical synthesis. 3. The question of whether these metabolites are true degradative intermediates is discussed, and a degradative pathway of compound (III) to the possible precursor of compound (VII), 7-carboxy-4-methyl-3,5-dioxoheptanoyl-CoA (IX), is tentatively proposed. The further degradation of compound (IX) to small fragments is also considered.


Comparative studies on glutamate metabolism in synpatic and non-synaptic rat brain mitochondria.

1. The apparent Michaelis constants of the glutamate dehydrogenase (EC 1.4.1.3), the glutamate-oxaloacetate transaminase (EC 2.6.1.1) and the glutaminase (EC 3.5.1.2) of rat brain mitochondria derived from non-synaptic (M) and synaptic (SM2) sources were studied. 2. The kinetics of oxygen uptake of both populations of mitochondria in the presence of a fixed concentration of malate and various concentrations of glutamate or glutamine were investigated. 3. In both mitochondrial populations, glutamate-supported respiration in the presence of 2.5 mM-malate appears to be biphasic, one system (B) having an apparent Km for glutamate of 0.25 +/- 0.04 mM (n=7) and the other (A) of 1.64 +/- 0.5 mM (n=7) [when corrected for low-Km process, Km=2.4 +/- 0.75 mM (n=7)]. Aspartate production in these experiments followed kinetics of a single process with an apparent Km for glutamate of 1.8-2 mM, approximating to the high-Km process. 4. Oxygen-uptake measurement with both mitochondrial populations in the presence of malate and various glutamate concentrations in which amino-oxyacetate was present showed kinetics approximating only to the low-Km process (apparent Km for glutamate approximately 0.2 mM). Similar experiments in the presence of glutamate alone showed kinetics approximating only to the high-Km process (apparent Km for glutamate approximately 1-1.3 mM). 5. Oxygen uptake supported by glutamine (0-3 mM) and malate (2.5 mM) by the free (M) mitochondrial population, however, showed single-phase kinetics with an apparent Km for glutamine of 0.28 mM. 6. Aspartate and 2-oxoglutarate accumulation was measured in 'free' nonsynaptic (M) brain mitochondria oxidizing various concentrations of glutamate at a fixed malate concentration. Over a 30-fold increase in glutamate concentration, the flux through the glutamate-oxaloacetate transaminase increased 7--8-fold, whereas the flux through 2-oxoglutarate dehydrogenase increased about 2.5-fold. 7. The biphasic kinetics of glutamate-supported respiration by brain mitochondria in the presence of malate are interpreted as reflecting this change in the relative fluxes through transamination and 2-oxoglutarate metabolism.


Effects of acetoacetate administration on glucose metabolism in mammary gland of fed lactating rats.

Administration of acetoactate to fed lactating rats rapidly decreases glucose uptake by the mammary gland, and causes an output of pyruvate, whereas lactate uptake remains unchanged. Similar changes, though not identical, occur in starved lactating rats, which suggests that the increased acetoacetate concentration in this situation may be one of the factors responsible for the alterations in glucose metabolism.


[Total dose determination at different dose rates in interstitial and endocavitary radiotherapy (author's transl)].

A method to determine biologically equivalent doses at different dose rates in interstitial and endocavitary radiotherapy is exposed. Doses may be directly determined by means of coefficients for single dose treatments and by means of N.S.D. concepts for fractionated schedules.


[Clinical and muscle istochemical observations in secondary hypokalaemia (author's transl)].

A case of hypokalaemia due to chronic administration of Clortalydone is reported. The histochemistry of muscle biopsy showed the morphologic changes which are usually found in the muscle fibers of periodic familial paralysis (necrotic fibers, accumulation of PAS positive substance, inflammatory cells, intermyofibrillar network degeneration, increase of lipids content). Such findings suggest some clues to the pathophysiology of the essential hypokalaemic paralysis and the possible practical importance of these histopathologic muscular findings in the diagnosis of secondary hypokalaemia.


[Antiphlogistic effects of extracts of a marine ooze in rats, guinea-pigs and rabbits (author's transl)].

Acetonic extracts of a marine ooze from the Adriatic coast inhibited in rats, and to a lesser extent in guinea-pigs and rabbits, the oedema-forming activity of histamine, but not of 5HT, bradykinin, formalin and carrageenin.


[Physiopathological and functional semeiologic considerations in a case of primary normoaldosteronemic hyperaldosteronism].

Among the atypical pictures of primary aldosteronism, sometimes, normal blood and urine concentration of aldosterone have been observed in association with an adrenal aldosterone-producing adenoma. Here we report a case of atypical primary aldosteronism so characterized: -- the patient had the typical clinical findings of aldosteronism (hypertension, hypokalemic alkalosis, polyuria, etc). -- the patient exhibted all the biochemical abnormalities of primary aldosteronism: increase of exchangeable Na and of plasma volume, decrease of exchangeable K, etc. -- the patient had normal blood and urine levels of aldosterone. -- the patient's blood and urine aldosterone concentration increased following sodium depletion and K administration. Such increase was comparable with that obtained in normal subjects after the same tests. However, at the end of these tests, the patient was still in potassium depletion and sodium repletion. Therefore, it was concluded that the secretion of aldosterone, although normal in absolute values, was inappropriate to the metabolic status of the patient, since such "normal" values were found in association with conditions that should have produced an inhibition of aldosterone production. The catheterization of adrenal veins demonstrated the existence of a right adrenal adenoma. The blood pressure and the biochemical parameters of the patients have been normalized by right adrenalectomy.


Effect of cholesterol feeding and estrogen treatment on synthesis of fatty acids in liver.

The effect of cholesterol feeding and estrogen administration on synthesis of fatty acids in liver mitochondria, microsomes and cytoplasm of male rabbits has been investigated. The synthesis was measured by the incorporation of [1(-14)C] acetyl CoA or [2(-14)C]malonyl CoA into long chain fatty acids under optimal conditions. It was found that atherogenesis markedly decreased the fatty acid synthesis in cytoplasm. The mitochondrial fatty acid synthesis was not affected by the disease. There was a small but measurable decrease in the synthesis of fatty acids in microsomes. Estrogen had no effect on the synthesis of fatty acids in mitochondria or microsomes. But if effectively counteracted, after a short lag period, the decreased synthesis of cytoplasmic fatty acids observed in atherosclerosis. It is possible that liver fatty acid synthetase is one of the enzyme systems through which estrogens exert their atherosclerosis-retarding effect. The decreased cytoplasmic fatty acid synthesis observed in atherosclerosis might account for the low levels of saturated fatty acids reported in liver and plasma lipids of atherosclerotic animals.


Aortic size and aortic calcification. A necropsy Study.

Measurements of aortic length and circumference in 336 post-mortem specimens confirm earlier, neglected observations on the progressive increase in aortic size which occurs with advancing years. The increase is not related to atherosclerosis , or to hypertension and seems to be part of a true ageing process. The value of measurement of aortic size in body age determination merits exploration by forensic pathologists. Aortic calcification is found in raised and complicated atherosclerotic plaques and its prevalence and severity closely follows the accepted pattern of plaque severity, occurring earlier and more severely in men, in the abdominal aorta and in patients with overt vascular disease in other territories such as patients with cardiac infarcts. No association was found between the amount of calcification and the presence of hypertension, diabetes or neoplasia.


Stimulation of aortic protein synthesis in experimental rabbit atherosclerosis.

Collagen, elastin and non-fibrous protein synthesis were measured in the aortas of male New Zealand white rabbits fed a diet containing 2% cholesterol for 140 or 180 days. At these time periods increases in aortic cholesterol and cholesteryl esters were evident. The atherosclerotic lesions induced were predominantly of the foam cell type although some areas of early fibrous lesion formation were noted. These changes in lipid concentration and arterial morphology were accompanied by a significant increase in collagen synthesis as determined by the formation of [14C]hydroxyproline. This increase, however, was not confined specifically to collagen since both elastin and non-collagenous proteins were also being synthesized at a higher rate. The two-fold increase in the rates of both fibrous and non-fibrous protein synthesis may in part be a consequence of marked intimal hyperplasia necessitating a general increase in protein synthesis.


Comparison of the lipid-lowering effect of clofibrate, and of clofibrate plus beta-pyridylcarbinol.

Forty-eight patients under 65 years were included in a double blind study comparing the lipid-lowering effect of clofibrate with that of beta-pyridylcarbinol combined with clofibrate. Over 4 months there was no significant difference in the lipid-lowering effect of either regime. A mean reduction of triglyceride of approximately 30% and of cholesterol of 18% was observed. Both drugs caused significantly greater reductions than placebo. No serious side-effects were noted.


The effect of treatment with clofibrate on hepatic triglyceride and lipoprotein lipase activities of post heparin plasma in male patients with hyperlipoproteinemia.

Twelve male patients with hyperlipoproteinemia were treated with clofibrate, 1 g twice daily. Serum triglyceride concentration decreased on the average 28 +/- 6%. No significant change of serum cholesterol concentration occurred. Post heparin plasma lipoprotein lipase activity isolated and partially purified by heparin Sepharose affinity chromatography was determined quantitatively. During the clofibrate treatment this enzyme activity increased 48 +/- 9%. The post heparin hepatic triglyceride lipase did not change significantly. The possibility that the serum triglyceride-lowering effect of clofibrate might partly be explained by an increased removal rate of triglyceride rich lipoproteins through increased lipoprotein lipase activity is discussed.


The effect of anticonvulsant drugs on thymidine and deoxyribosenucleic acid synthesis by human marrow cells.

An abnormal deoxyuridine suppression test was found in treated epileptic patients that did not correlate with other evidence of abnormal folate metabolism. Diphenylhydantoin at a level of 100 microgram/ml interfered with the incorporation of [3H]thymidine into DNA. It is suggested that anticonvulsants may exert their effect at a stage of DNA synthesis beyond that at which folate coenzymes operate. Folate deficiency may then arise as a result of death of cells in vivo causing an increased folate requirement.


Echocardiographic findings in left ventricular to right atrial shunts.

The echocardiographic abnormalities of tricuspid valve motion in 2 patients with left ventricular to right atrial shunts are described. In both patients the abnormal anatomy was defined at surgery, in one patient the shunt being above the tricuspid valve leaflets (supravalvar) and in the other patient through the septal leaflet (intravalvar). Different patterns of tricuspid valve systolic fluttering were seen in these two cases and the possible reasons for this are discussed. After surgical closure of the defects the systolic fluttering of the tricuspid valve was no longer observed. Echocardiography appears to be useful in detecting the presence of left ventricular to right atrial shunts which otherwise may be difficult to diagnose.


Intra-aortic balloon assistance in cardiogenic shock after myocardial infarction or cardiac surgery.

Sixty-eight patients were referred for consideration of intra-aortic balloon assistance, 55 of whom were accepted. Thirty-one patients were in cardiogenic shock after myocardial infarction and the remaining 24 were cardiac surgical patients. Twenty-three of the myocardial infarct group were established on IABA and all 24 of the cardiac surgical patients. Of the 23 patients with cardiogenic shock after myocardial infarction, 19 showed initial haemodynamic improvement on intra-aortic balloon assistance and 5 (22%) survived to leave hospital. Of the 24 cardiac surgical patients, 15 could not be withdrawn from total cardiopulmonary bypass. With intra-aortic balloon assistance, 11 (73%) could be withdrawn from cardiopulmonary bypass and 5 (33%) were hospital and long-term survivors. The remaining 9 surgical patients were in cardiogenic shock in the early postoperative phase, though 5 showed initial haemodynamic improvement there was only one hospital survivor in this group. Intra-aortic balloon assistance was, therefore, of most value in patients dependent on cardiopulmonary bypass. The survival in patients with cardiogenic shock after myocardial infarction was marginally improved.


Infradiaphragmatic total anomalous pulmonary venous return. Review of clinical and pathological findings and results of operation in 28 cases.

Twenty-eight cases of infradiaphragmatic total anomalous pulmonary venous return are presented, 17 without associated complex intracardiac anomalies (group A), and 11 with additional complex lesions (group B). The anomalous site of connection was to the portal vein in 19 cases (68%), to the inferior vena cava in 4 (14%), the ductus venosus in 2 (7%), to the left hepatic vein in 2 (7%), and unknown in one. A patent foramen ovale was present in 82 per cent of cases in group A and 40 per cent in group B and was frequently associated with a small left atrium and left ventricle. Nine cases (8 in group A; 1 in group B) had surgical correction, with 3 long-term survivors. The surgical mortality was 66 per cent. The postoperative haemodynamic status of the 3 surviving patients is very satisfactory, though 1 had a residual atrial septal defect. Factors which adversely affected the surgical outcome were: (1) a critically ill infant, (2) small left atrium and left ventricle, (3) a patent foramen ovale rather than atrial septal defect, (4) systemic arterial oxygen saturation less than 70 per cent, and (5) pulmonary arterial pressure in excess of systemic arterial pressure. The mortality for the entire series was 93 per cent.


Sequence of retrograde atrial activation of the human heart. Correlation with P wave polarity.

Studies were performed in 6 patients during open heart surgery to correlate the relative sequence of atrial activation with the polarity of the retrograde P wave in electrocardiographic leads II, III, and aVF. Bipolar atrial electrograms were recorded from selected sites during threshold pacing from sites low on the right side of the atrial septum which when paced resulted in the inscription of either negative or positive P waves in electrocardiograph leads II, III, and aVF. When the atria were paced from a site just anterior to the coronary sinus ostium (negative P waves), the posteroinferior left atrium was activated early and Bachmann's bundle late during the inscription of the P wave. When the atria were paced from a site 2 cm anterior to the coronary sinus ostial site (positive P waves), Bachmann's bundle was activated early during the P wave and the posteroinferior left atrium was activated relatively later. These data from man are consistent with previous studies in the canine heart. Therefore, we deduced that in man during the inscription of negative retrograde P waves in leads II, III, and aVF, atrial activation occurs primarily in a retrograde fashion. However, during the inscription of positive retrograde P waves in man, activation occurs rapidly up the  interatrial septum (we believe via the anterior internodal pathway) to Bachmann's bundle, from where it then spreads in a manner similar to that which occurs during normal sinus rhythm.


Dual effect of disopyramide on atrial and atrioventricular conduction and refractory periods.

Disopyramide has been shown in conditions of cholinergic blockade to have a depressant effect upon sinus node automaticity and the atrial refractoriness. It also prolongs atrioventricular conduction and increases atrioventricular refractoriness. These effects may often be masked in vivo by the anticholinergic effects of the drug.


Dissimilar atrial rhythms. A patient with interatrial block.

A patient with type A Wolff-Parkinson-White syndrome and prolonged interatrial conduction intervals developed atrial flutter during the course of an electrophysiological study. The atrial flutter blocked along the left-to-right conduction pathways in a Wenckebach pattern. The dissimilar atrial rhythms of right atrial tachycardia and left atrial flutter evolved as the interatrial block increased to 2:1 conduction.


Infradiaphragmatic total anomalous pulmonary venous connection to portal vein. Diagnostic implications of echocardiography.

A case of total anomalous pulmonary venous connection to the portal vein is described. The diagnosis was suspected clinically, supported by the echocardiogram, and confirmed by cardiac catheterisation, angiocardiography, and contrast echocardiography. An echo-free space lying behind the left atrium initially was thought to represent the common pulmonary vein. However, contrast echocardiography showed that this space was not the anomalous vein but probably an artefact. This paper shows that the origins of intracardiac echoes cannot always be assumed from a simple comparison of echocardiography with angiocardiographic or necropsy findings. In some cases it is necessary to introduce a marker into the echocardiogram which unequivocally originates from, and, therefore, localises, the structure under examination. Contrast echocardiography provides such a marker.


Vasodilator treatment for acute and chronic heart failure.

The current status of the use of vasodilator drugs in the treatment of acute and chronic heart failure has been reviewed. It is apparent that vasodilator treatment can be used effectively in some patients with heart failure with a beneficial haemodynamics response, and that vasodilator agents are likely to find an important place in the management of such patients. Vasodilator treatment may be associated with complications and must be used with care. Though several nonparenteral vasodilator agents have been investigated, no ideal drug is yet available for the treatment of chronic heart failure. Nevertheless, it is probable that suitable drugs will emerge and find an important place in the management of such patients.


Evaluation of intracardiac recordings in diagnosis of impulse formation and concealed conduction in atrioventricular nodal bypass tracts.

Invasive electrophysiological studies were performed in 2 symptomatic patients with recurrent arrhythmias in which impulse formation presumably occured within atrioventricular nodal bypass tracts. Case 1 had ectopic beats arising within, or close to, the upper end of a left-sided atrioventricular nodal bypass tract of the type described by Brechenmacher. In addition, this conduction was 'concealed' during sinus rhythm and right atrial pacing because the relatively prolonged right-to-left atrial conduction time allowed right atrial impulses to reach the His bundle via the atrioventricular node before they could do so through the atrioventricular nodal bypass tract. Case 2 had ectopic beats arising in a right-sided atrioventricular nodal bypass tract which did not conduct in either forward or retrograde directions, its presence being detected only when initiating impulses. However, it could not be determined whether this tract was an 'abnormal' atrio-His connection or a 'normal' transitional (atrio-atrioventricular nodal) tract. Though intracardiac studies complement body surface recordings, they should be interpreted with knowledge of their inherent limitations.


Early work load tests for evaluation of long-term prognosis of acute myocardial infarction.

Exercise tests performed 3 and 9 weeks after acute myocardial infarction in 205 patients were found to give prognostic information on the survival during a follow-up period of 2 to 5 years. The appearance of tachycardia, major ventricular arrhythmias, or anginal complaints during these early exercise tests was thus accompanied by a significantly increased mortality during the observation period. Ventricular arrhythmias disclosed by exercise proved to be of higher prognostic significance than those recorded at rest on the same occasions. The usefulness of early exercise tests in the evaluation of the response to antiarrhythmic treatment after acute myocardial infarction as well as of the prognostic importance of the effects was documented in a smaller series of patients.


Indirect measurement of sinoatrial conduction time in patients with sinoatrial disease and in controls.

Clinical recognition of sinoatrial disease currently depends on the presence of transient sinus bradycardia, sinoatrial block, or supraventricular tachyarrhythmias. The value of clinical electrophysiological assessment in these patients is not clear. Using intracardiac electrophysiological recordings and programmed stimulation we have examined 14 patients with sinoatrial disease and 11 control patients undergoing investigation for chest pain. Intracardiac conduction times were normal in all patients. There was no significant difference of sinus node recovery times between the sinoatrial disease and control groups. Sinoatrial conduction times were measured by the indirect method and two populations were identified. However, the mean values of 128 +/- 27 ms in patients and 112 +/- 30 ms in controls were not significantly different and major overlap rendered this measurement clinically valueless. It is concluded that no current electrophysiological measurement has diagnostic value in patients with sinoatrial disease.


The Elag-Koln automatic blood pressure recorder. A clinical appraisal.

The performance of an Elag-Koln semiautomatic blood pressure recorder was compared with the London School of Hygiene mercury sphygmomanometer in 24 subjects providing a wide range of blood pressure measurements. Readings with the two instruments correlated highly (for systole, r=0-99; for diastole phase 4, r=0-97; for diastole phase 5,r=0-98), and the slopes of the regressions did not differ significantly from unity. Elag-Koln measurements were higher for systole (mean difference 3-7 mmHg,P less than 0-001) and diastole phase 5 (mean difference 7-4 mmHg,P less than 0-001), but agreed closely with diastole phase 4 readings with the London School of Hygiene instrument. The Elag-Koln recorder tested was compact, easy to use, and had acceptable accuracy. This type of instrument deserves further testing to examine its suitability for general use.


Baroreflex sensitivity and cardiopulmonary blood volume in normotensive and hypertensive patients.

Baroreflex sensitivity and cardiopulmonary blood volume were determined in 95 men, including normotensive and hypertensive subjects with normal renal function and balanced sodium intake and urinary output. Baroreflex sensitivity was estimated by determining the slope of the regression line relating the increase of systolic pressure to the cardiac slowing after transient rises of arterial pressure. A technique of gradual atropinisation was used to evaluate the parasympathetic mediated component of the reflex. With this method, it was possible to calculate the exact atropine dose abolishing the reflex sensitivity. This index was not dependent on age. It was negatively correlated to the diastolic pressure in normotensive patients but not in hypertensive patients. The ratio between the cardiopulmonary and the total blood volume was considered as an index of sympathetic venous tone. This ratio was positively correlated to the diastolic pressure in normotensive patients, but not in hypertensive patients. This study strongly suggests that a precise sympathetic-parasympathetic balance existed in the normotensive patients. This balance was disrupted in the hypertensive patients pointing to abnormalities in the autonomic nervous system of permanently hypertensive patients.


Left ventricular apical thin point.

The wall thickness of the apex of the left ventricle has been measured at its thinnest point in 60 adult hearts at necropsy. This measured 2 mm or less in 97% of them and 1 mm or less in 67%.


Atrioventricular reciprocal rhythm and chronic reciprocating tachycardia in a newborn infant with concealed Wolff-Parkinson-White syndrome.

A case of atrioventricular reciprocal rhythm and chronic reciprocating tachycardia in a newborn infant is presented. Electrophysiological studies suggest that these rhythm disturbances are related to the presence of a right-sided atrioventricular accessory pathway capable only of retrograde conduction (concealed Wolff-Parkinson-White syndrome). The technique of recording the sequence of atrial activation during the tachycardia is described and its clinical importance emphasised.


Primary thrombosis of pulmonary artery in a child with tetralogy of Fallot.

A 34-month-old child with tetralogy of Fallot developed an acute, primary thrombosis of the pulmonary trunk. A greatly increased haematocrit was most probably a predisposing factor. An unusual postmortem finding was the large number of extremely dilated myocardial capillaries. In a review of a series of 394 consecutive necropsy cases with many kinds of cardiac anomalies, there was only one case (a 28-year-old man) with occluding thrombus formation in the pulmonary artery. This case also belonged to the Fallot group but a subacute glomerulonephritis seemed to be the major cause of death.


Kinetics of chlorophyll fluorescence at 77K in Chlorella and chloroplasts. Effects of CCCP, ferricyanide and DCMU.

The kinetics of chlorophyll fluorescence at 77K were studied in Chlorella cells and spinach chloroplasts. During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation. The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) both in Chlorella and chloroplasts. The fluorescence rise during a second illumination is still biphasic. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts. Ferricyanide which oxidizes cytochrome beta-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination. Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.


Phase separation in phosphatidylcholine bilayers as a predictor of inhibition of blood platelet aggregation by amantadines.

The ability of eleven amantadine derivatives to induce phase separation in dipalmitoyl phosphatidylcholine bilayers was studied by differential scanning calorimetry. The relative potency varied with the shape and size of the hydrocarbon cage. These agents also markedly inhibited blood platelet aggregation. The relative potencies of these compounds to induce phase separation showed a significant correlation (r = 0.70) with their platelet inhibitory activity suggesting that their pharmacologic action may be at the level of the platelet membrane. The effective concentration of the parent component amantadine is similar to its pharmacologic concentration suggesting its use as an anti-platelet drug.


Interaction between sphingomyelin and a cytolysin from the sea anemone Stoichactis helianthus.

The cytolytic toxin from the sea anemone Stoichactis helianthus was inhibited up to 90--95% by suspensions of sphingomyelin but not by phosphatidylcholine or other membrane lipids. When the toxin was incubated with sphingomyelin and the mixture fractionated either by isoelectric focusing or Sephadex gel filtration, the residual hemolytic units migrated together with the lipid and not as free toxin. Incubation with phosphatidylcholine, however, did not shift the toxin peak in either type of column. A toxin-ferritin conjugate retaining hemolytic activity was observed by negative staining to bind to liposomes prepared with sphingomyelin but not with liposomes containing phosphatidylcholine. The results provide evidence that the membrane binding site of the toxin is sphingomyelin.


Calcium-induced interaction of phospholipid vesicles and bilayer lipid membranes.

The interaction of unilamellar phospholipid vesicles with bilayer lipid membranes has been studied by observing the electrical conductance of the planar membrane. The presence of phosphatidylcholine vesicles as well as phosphatidylcholine/phosphatidylserine (1 : 1) vesicles on one side compartment of the bilayer membrane, but not phosphatidylserine vesicles, causes discrete fluctuations in the phosphatidylserine membrane conductance, which is also increased by at least an order of magnitude. These events are dependent on vesicle concentration as well as the presence of Ca2+. The results are interpreted in terms of the incorporation of domains of phosphatidylcholine into the membrane, which confer a higher conductance state.


Biosynthesis of brush border glycoproteins by human small intestinal mucosa in organ culture.

The incorporation of [14C]glucosamine into brush border glycoproteins by human small intestinal mucosa in organ culture has been investigated. The experiments were based on the observations that (1) isolated brush border membrane fragments from cultured explants showed an unchanged pattern of protein bands and brush border enzyme activities on sodium dodecyl sulfate/polyacrylamide gels after electrophoresis and (2) the rate of overall [14C]glucosamine incorporation measured in the tissue homogenate remained constant up to 48 h. After 24 h of culture, the radioactivity peaks on gels due to incorporation of [14C]glucosamine were found exclusively in the high molecular weight region and corresponded to protein bands identified as maltase-glucoamylase, lactase, sucrase-isomaltase, enterokinase and alkaline phosphatase. Enzymatic activity could not be assigned to the three remaining labelled bands. Most of these glycoproteins were already labelled after 5 h. Newly glycosylated brush border enzymes remained predominantly associated with the brush border membrane of intact cells with little release into the medium up to 24 h.


The chloride transport induced by triaklyl-tin compound across erythrocyte membrane.

The effect of tripropyl-tin chloride on anion permeability was studied using red cells previously treated with a covalent binding inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to inhibit completely and irreversibly the natural anion transport system. It was demonstrated that the tin compound can mediate chloride-hydroxide and chloride-chloride exchanges across the "impermeabilised" erythrocyte membrane. In the non hemolytic range, the rate of exchange increased with the concentration of the tin compound in a non linear fashion, and no saturation effect was seen. The temperature profile of the chloride self exchange induced by tripropyl-tin was studied and the apparent activation energy found was 29 Kcal/mol. The tripropyl-tin chloride cannot mediate a chloride-bicarbonate exchange. Because of this discriminatory effect between hydroxide and bicarbonate, the tin compound can be useful in certain experimental conditions as seen for the study of the anion "carrier" of the red cell membrane ("cousin, J.L., Motais, R. and Sola, F. (1975) J. Physiol. Lond. 253, 385-399).


Stimulatory and inhibitory effects of sulfhydryl reagent on p-aminohippuric acid transport by isolated renal tubules.

Isolated tubules from rabbit kidney cortex were treated with several different sulfhydryl reagents in an attempt to determine whether sulfhydryl groups are involved in organic acid transport. Disulfide reagents such as sodium tetrathionate and 6,6'-dithionicotinic acid were found to exert a biphasic effect on p-aminohippuric acid transport, i.e. transient stimulation followed by inhibition. In contrast, treatment of tubules with the mercaptide-forming reagent, p-chloromercuribenzoate, caused only inhibition of organic acid transport. Treatment of tubules with reductants such as dithiothreitol or mercaptoethanol blocked the stimulatory effect of tetrathionate without affecting the inhibitory effect of this oxidant. The inhibition caused by p-chloromercuribenzoate, however, was largely reversible when tubules were treated with reductants. The results suggest that the renal organic acid transport system contains sulfhydryl groups and that its activity is increased when some of these groups are oxidized.


How the kinetic parameters of the simple carrier are affected by an applied voltage.

The kinetic parameters (K, R00, R12, R21 and Ree) of the simple carrier model are analysed as a function of applied voltage for various cases of charged substrates and carriers. If certain parameters are invariant with voltage, strong conclusions can be made as to the charge on the free carrier. In one particular case (where the parameter R00 is invariant while R12 and R21 display a non-linear dependence on the exponential of the voltage) it may be possible to identify the isomerisation of the carrier-substrate complex.


Phase behavior and structural characteristics of hydrated bovine brain gangliosides.

Hydrated bovine brain gangliosides have been studied by differential scanning calorimetry, X-ray diffraction, and polarized light microscopy. Over the hydration range 18-50 wt.% H2O, mixed brain gangliosides exhibit a hexagonal mesophase structure, in which the ganglioside molecules form hexagonally packed rod-like structures. The apolar lipid chains radiate from the center of the rods, with the sugar groups on the cylinder surface in contact with water. At higher water contents, an isotropic micellar solution is formed. Over the hydration range 20-30 wt.% H2O, two small thermal transitions with peak maxima at 30 degrees C and 46 degrees C are observed by differential scanning calorimetry. These transitions broaden and move apart in temperature as the hydration is increased to 50 wt.% H2O. X-ray diffraction data indicate that this double transition is associated with a hydrocarbon chain rearrangement from a disordered state to another, possibly more disordered, state. Thus, the gangliosides, although membrane lipid components, have physical characteristics which are very different from those of the membrane phospholipids.


The computation of saturable and linear components of intestinal and other transport kinetics.

1. Published data for absorption kinetics have been fitted by non-linear regression to (i) a single Michaelis-Menten function, (ii) a Michaelis-Menten function plus a linear term and (iii) a sum of two Michaelis-Menten functions. A series of criteria have been drawn up to establish the goodness of fit in each case. 2. In 17 out of 35 cases the Michaelis-Menten function was the "best fit". In nine cases the "best-fit" model also included a linear term, but never was the sum of two Michaelis-Menten functions accepted to be the "best-fit" model. 3. Linearity of a Lineweaver-Burk plot was of unreliable diagnostic value in assessing goodness of fit. 4. Since the fit of a Michaelis-Menten function was often poor, simulated data sets with error were used to study the influence of experimental design etc. on Michaelis-Menten parameter estimation. 5. Precision of estimation of Km is increased by increasing the number of data points, reducing their variance, increasing the data range and by straddling Km in the observations. For a given constant number of observations there is no advantage in using replicate observations at few concentrations or single values at relatively many concentrations, or in using single values rather than means. 6. The caution necessary in interpretation of kinetic models is emphasized.


Intrinsic proteins of the intestinal microvillus membrane. Iodonaphthylazide labeling studies.

Isolated brush border membranes of the intestinal epithelial cell were labeled with a hydrophobic photoactive compound [125U]iodonaphthylazide. High incorporation of the radioactive naphthylazide was noted for molecular weight bands of 99 000, 86 000, 65 000, 54 000 and 30 000. Minimal labeling occurred in the higher bands of 300 000, 135 000, 125 000 and 17 000. The iodonaphthylazide label was not removed by extensive papain digestion whereas chloramine T iodinated membranes released radioactivity under the same conditions. Neither enzymatic nor transport activities were inhibited by the presence of iodonaphthylazide or the irradiation process. On the basis of the presented data it is concluded that the iodonaphthylazide unspecifically labels those portions of membrane proteins which are inserted into the lipid bilayer matrix.


Permeability of iodide in multilamellar liposomes modeled by two compartments and a reservoir.

A previously published rate law for the diffusion of iodide from multilamellar egg phosphatidylcholine liposomes (Schullery, S.E. (1975) Chem. Phys. Lipids 14, 49-58) is fitted to the relatively simple mathematical model of two compartments in series with a reservoir. All of the inner liposome compartments are assumed to behave as effectively one compartment in series with the liposome's outermost compartment. Based on this model, reasonable values are calculated for the fraction of the total solution trapped by liposomes which is in the outermost liposome compartment, 17%, and the permeability coefficient of iodide against isotonic, mixed iodide-chloride solution, 2-10(-9) cm/s.


Identification of transferrin receptors in reticulocytes.

Experiments were performed to obtain definitive evidence for the presence of membrane receptors for transferrin on reticulocytes. Rabbit reticulocytes were incubated with 125I-labelled rabbit transferrin. The transferrin taken up by the cells was solubilized using the non-ionic detergent, Teric 12A9 (polyoxyethylene (n=9) dodecyl alcohol). The soluble extracts of the cells were examined by gel filtration and a transferrin-binding moiety of approximate molecular weight 275 000 was identified. This binding moiety was found only in reticulocytes, not in mature erythrocytes. The membrane component could bind only transferrin and not the other plasma proteins studied. Only transferrin could displace bound transferrin from the complex. Rabbit transferrin was bound more strongly than human transferrin. The binding of transferrin to the component was shown to be reversible and saturable. It is concluded from these studies that the transferrin binding component identified in the reticulocyte stroma is a true physiological receptor for transferrin.


Effect of bicarbonate and other buffers on choroid plexus Na+/K+pump.

The Na+/K+ pump on the apical membrane of the choroid plexus epithelium was found to be sensitive to bicarbonate. Glycodiazine, a non-volatile, lipid soluble buffer with a pK of 5.7, mimicked the effect of bicarbonate, and was transported across the epithelium in the same direction as sodium. These results are explained in terms of a Na+/H+ exchange mechanism on the basal-lateral membrane.


Cooperative unit size in the gel-liquid crystalline phase transition of dipalmitoyl phosphatidylcholine-water multilayers: an estimate from Raman spectroscopy.

The temperature dependence of the Raman spectral transitions assigned to the acyl chain C-C stretching modes of dipalmitoyl phosphatidylcholine was determined for the gel, phase transition and liquid crystalline states of the lipid multilayers. The van't Hoff enthalpy differences delta HVH between trans and gauche rotational isomers were obtained from the Raman spectral data for the temperature region characteristic of each bilayer state. An average size for the cooperative unit undergoing the chain melting process during the phase transition was estimated from the ratio of the appropriate van't Hoff enthalpy to an adjusted calorimetric enthalpy.


An electron microscopic histochemical investigation of the localization of creatine phosphokinase in heart cells.

The results of an electron microscopic histochemical investigation performed in the current study indicate that in heart cells creatine phosphokinase is localized: (1) inside mitochondria mainly on the cristae membranes, (2) on the membrane of the sarcoplasmic reticulum, (3) on myofibrils (and in cytoplasm), (4) on the plasma membrane of the cells, (5) on the membrane of the cell nuclei.


Conformation affinity of cross-linked polyacrylic gels towards nucleobases, nucleosides and nucleotides.

By radical copolymerisation of monofunctional acrylic derivatives with 1,4-tetramethylene dimethacrylate, cross-linked polyacrylic gels which show a high affinity towards nucleic acid residues have been synthesised. Using these polyacrlic gels, mixtures of nucleobases, nucleosides and nucleotides can be chromatographically separated to differing extents, in some cases quantitatively. The elution of nucleobases and nucleosides from the gel in the order Cyt, Gua, Thy, Ade (or dC, dG, dT, dA and C, G, U, A, respectively) shows that Ade (dA, A) is retarded to the highest degree from the gel matrix, Cyt (dC, C) to the lowest degree. Further, the results of the separations prove that the affinity of the polyacrylic gels is much stronger towards deoxyribonucleosides than towards ribonucleosides and nucleobases. The affinity of the polyacrylic gels towards nucleobases and nucleosides depends mainly upon their tertiary structure whereas the functional side groups of the polyacrylate matrix do not contribute significantly to the interaction of the gels.


The incomplete denaturation of DNA in N,N-dimethyl formamide.

In this paper we show that the extent of DNA denaturation achieved by dialysis against N,N-dimethyl formamide depends on both the source of DNA and the buffer used. The stability against denaturation increases with the guanine plus cytosine content. The base sequence also plays a minor role. This behavior is due to differences in the solubility of DNA in the denaturing solvent. The DNAs with a high guanine-cytosine content have a lower solubility in dimethyl formamide and this fact results in a higher stability against denaturation. As a practical consequence, in order to achieve complete denaturation of DNA by dialysis against dimethyl formamide, it is necessary to start with DNA dissolved in a buffer of low ionic strength, preferably below 10(-3) M.


Control of cell protein degradation. Changes in activities of lysosomal proteases.

In cycloheximide-treated fibroblasts, the rate of cell proteolysis and the specific activity of cathepsin D show a rapid, concurrent, and proportional decrease over a 48 h period. Cathepsin B1 also decreases, but other lysosomal enzymes show litte or no change in specific activity. These findings are consistent with the hypothesis that the rate of proteolysis in the cell is in part controlled by the specific activities of lysosomal proteases.


C8-amino purine nucleosides. A well-defined steric determinant of glycosyl conformational preferences.

100 MHz proton magnetic resonance measurements were performed on dilute solutions of adenosine and guanosine and their 8MH2, 8-NHCH3 8-N(CH3)2 and 8-bromo derivatives. The chemical shift of the ribose C2'-H and especially the difference in chemical shifts between the C1'-H and C2'-H resonances clearly indicated whether the nucleoside exists in a syn glycosyl conformation (the C8-dimethylamino derivatives) or as a flexible syn-anti mixture (the monomethylamino and amino derivatives). The temperature dependent behavior of these indicators can be employed to define qualitative shifs in syn-anti equilibrium with temperature. An increased C1'-H-C2'-H chemical shift separation implies shift to more anti, a decreased separation a shift to more syn conformers.


Bromodeoxyuridine inhibition of Friend leukemia cell induction. Mechanism of reversal by deoxycytidine.

Deoxycytidine (dC) reverses bromodeoxyuridine (brdUrd) inhibition of induction in Friend leukemia cells only if added within the first 6 h after the addition of brdUrd. dC is shown to reduce the uptake of [3H]brdUrd into both soluble nucleotide pools and DNA, substantially expand the dTTP pool, and result in a lower level of brdUrd substitution in DNA. When the conversion of dC to thymidine nucleotides is prevented in BATH medium (containing brdUrd, aminopterin, thymidine, and hypoxanthine), dC no longer reverses brdUrd inhibition. These results show that dC exerts its primary effect via alterations in thymidine pools and probably through the resultant lower substitution of brdUrd in DNA.


An efficient synthesis of polyguanylic acid by a thermophilic polynucleotide phosphorylase.

Polyguanylic acid (poly(G)) was synthesized from GDP in a yield of 60-75% by Thermus thermophilus polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) at 70 degrees C, pH 8.5 in the presence of Mg2+. The yield was dependent on the ratio of GDP to Mg2+, but was independent of the concentrations of enzyme or substrate. The maximal rate of GDP polymerization was obtained when the ratio of GDP to Mg2+ was 3:1. However, by prolonged incubation, the higher initial ratio of over 4:1 was preferred because of the rapid consumption of GDP in the reaction mixture. Poly(G) prepared by 1 h incubation was heterogeneous in size from 5 S to over 23 S, but by prolonged incubation of 19 h the size of product converged to 9 S as judged by sucrose density gradient centrifugation. Its chain length was determined by terminal nucleoside analysis to be 200 nucleotides long.


Purification and properties of tRNA nucleotidyl transferase from Musca domestica.

The enzyme tRNA nucleotidyl transferase (EC 2.7.7.25) has been highly purified from whole adult houseflies. A molecular weight of 30 000 has been determined. The enzyme requires Mg2+ and tRNA deprived of the 3' terminal sequence CCA for activity in the incorporation of AMP and CMP onto the tRNA. UMP can be incorporated instead of CMP but the latter has a higher affinity than UMP as shown by competition experiments. A complex between the enzyme and tRNA has been shown by sucrose gradient centrifugation, nitrocellulose binding and protection by tRNA against enzyme denaturation at 50 degrees C. Comparative studies with tRNA nucleotidyl transferase purified from larvae, pupae and adult insects indicate that tRNA nucleotidyl transferase from these three developmental stages have the same molecular weight, sedimentation coefficient and optimum pH while the larval enzyme differs from the pupae and adult enzymes in the elution behaviour from a DEAE-cellulose column.


Multiple chromatographic peaks of phenylalanyl-tRNA associated with spontaneous hydrolysis of Y base during isolation.

In contrast to the single phenylalanyl-tRNA found in normal cells, some tumours are known to have more than one phenylalanine isoacceptor. However, during certain steps in tRNA isolation from normal or tumour tissue, additional chromatographic peaks can be artificially produced which may be confused with the tumour-specific Phe-tRNA. Such procedures as extraction with unbuffered phenol and unbuffered gel filtration chromatography appear to produce adventitious isoacceptors by hydrolysis of Y base.


Effect of magnesium and polyamines on the structure of yeast tRNAPhe.

The effect of magnesium and polyamines (spermine, spermidine, putrescine and cadaverine) on the structure of yeast tRNAPhe has been investigated. It is found that magnesium induces structural changes and stabilizes hydrogen bonds in the temperature range 22--44 degrees C in 0.17 M sodium. The number of Mg2+ which affect tRNA structure increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C and the number of additional base pairs formed in the presence of magnesium increases from 1 +/- 1 at 22 degrees C to 4 +/- 1 at 44 degrees C. The spectral changes are more-or-less sequential. The polyamine spermine stabilizes some, but not all, of the structural features stabilized by magnesium at 44 degrees C, and the combination of magnesium and spermine, at low levels, is more effective than either cation alone in stabilizing tRNA structure. Comparison of the effects of spermine, spermidine, putrescine and cadaverine indicates that it is the asymmetric triamine unit which is important in the stabilization. Some spectral changes induced by magnesium can be assigned to stabilization of specific tertiary structure interactions and to alteration of stacking adjacent to U8-A14.


HeLa cell DNA polymerases: the effect of cycloheximide in vivo and detection of a new form of DNA polymerase alpha.

Blockage of protein synthesis in HeLa cells by cycloheximide leads to selective effects on the levels of DNA polymerases alpha, beta, and gamma in the cell. The total activity of DNA polymerase alpha remains unchanged after 7 h exposure of cells to cycloheximide but drops to 50% of its original level after 24 h. The level of the beta-polymerase falls rapidly in the cell and is reduced to less than 30% of its initial value by 7 h after treatment of the cells with cycloheximide. The gamma-polymerase level is diminished by 30--40% during the 7 h cycloheximide treatment and reaches 50% of its original level after 24 h. Cells which have been exposed to cycloheximide for 7 h will regain normal levels of the beta- and gamma-polymerases within 90 min after removal of the drug. The cycloheximide-treated cells also show the presence of a new form of the alpha-polymerase, designated alpha1, which can be clearly detected as a separate entity in column chromatography. The level of alpha1 in the nucleus increases during the period that the cells are treated and cycloheximide so that after 24 h it represents almost 50% of the nuclear DNA polymerase activity. The presence of alpha1 in the cytoplasmic fraction can also be demonstrated in both cycloheximide-treated and normal, growing cells.


Normal endonuclease activities for damaged DNA during hepatocarcinogenesis.

Two endonuclease activities in rat liver for damaged DNA were assayed. Double-stranded, covalently closed DNA from phage PM2 was damaged by either ultraviolet irradiation or by heating at acid pH, and used as substrate for endonucleases specific for ultraviolet DNA damage and for DNA apurinic sites, respectively. The levels of both enzyme activities in livers of normal rats were compared to levels in livers of rats fed N-2-acetylaminofluorene. At critical stages of the carcinogenic regimen levels of both endonuclease activities were normal. This, together with other data, suggests that depression of excision-repair of DNA damage does not take place during experimental carcinogenesis.


Stimulation of hepatocellular proliferation by a serum factor from thioacetamide-treated rats.

Rats treated with thioacetamide undergo hepatocellular proliferation reminscent of liver regeneration following partial hepatectomy. 36 h after administration of 50 mg thioacetamide/kg body weight to rats, [3H]thymidine incorporation into hepatic DNA reaches a peak of 78-10(3) dpm/mg DNA compared to a control of 3.2-10(3) dpm/mg DNA. Serum obtained from 6 to 48 h after administration of thioacetamide to rats stimulated hepatic but not kidney DNA synthesis in mice and rats. Autoradiography revealed an increase in the incorporation of labelled thymidine into the nuclei of mouse hepatocytes. The mitotic index of the liver was also increased. The serum factor stimulating these changes in the liver was non-dialyzable and heat stable. These results indicate that thioacetamide induced liver injury results in a humoral factor which stimulates DNA synthesis in rat and mouse identified in the serum from partially hepatectomized rats.


Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase.

Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacrylamide gel analysis of the purified acidic proteins and 80-S particles showed identical phosphoproteins in the 16 000 dalton region.


X-ray structure of a compound of cobalt with uridine 5'-monophosphate. Evidence for metal-phosphate bonding only.

An X-ray study of the crystal structure of a cobalt(II) complex with uridine 5'-monophosphate shows it to contain polymeric chains of composition [Co2(H2O)4 (5'-UMP)2]n. The cobalt atoms do not bind to the pyrimidine base moiety; instead each metal atom is octahedrally coordinated to four phosphate groups and two water molecules. Managanese(II) forms a similar compound.


Isolation and characterization of mRNA from Paramecium aurelia.

Total cellular RNA was isolated from the ciliate protozoan Paramecium aurelia by pH 9.5 chloroform/octanol extraction. Passage of this RNA through an oligo(dT)-cellulose column in 0.5 M NaCl resulted in 2--3% binding, indicating the presence of polyadenylic acid sequences. These polyadenylic acid regions were estimated to be 250-500 nucleotides in length, based on their resistance to ribonuclease degradation. The oligo(dT)-cellulose bound RNA sedimented at 14--25 S in sodium dodecyl sulphate/sucrose gradients. The base composition of this RNA is similar to the base composition of the DNA. This RNA was also actively translated into protein by an in vitro protein synthesizing system isolated from wheat germ. Translation was optimal under conditions similar to those used for mammalian mRNA translation. In addition, translation of the P. aurelia oligo(dT)-cellulose bound RNA was inhibited 80% by the analog 7-methylguanosine-5'-phosphate, suggesting the presence of a 5'-capped terminus.


DNA synthesis in isolated lymphocyte nuclei. Effects of megaloblastic anaemia due to folate or vitamin B-12 deficiency or antimetabolite drugs.

DNA synthesis has been studied in nuclei isolated from phytohaemagglutinin-stimulated lymphocytes from normal subjects and patients with megaloblastic anaemia. Lymphocytes were incubated for 72 h, nuclei isolated and incorporation of tritiated deoxythymidine triphosphate ([3H]TTP) into DNA measured, usually over a 10 min incubation period. Preincubation of normal phytohaemagglutinin-stimulated lymphocytes with methotrexate (1 - 10(-5) M, 48--72 h), 5-fluorouracil (1 - 10(-6) M, 70--72 h), and 1-beta-D-arabinofuranosyl cytosine (cytosine arabinoside) (4 - 10(-5) M, 71--72 h) caused a mean rise in [3H]TTP incorporation of 1.7 (P less than 0.01), 1.7 (P less than 0.05) and 2.4 (P less than 0.0025) fold, respectively. Hydroxyurea (3 - 10(-4) M, 48--72 h) in two experiments caused a mean increase of 1.6 fold. Untreated vitamin B-12- and folate-deficient cells showed a 2.0-fold (P less than 0.05) increase above the incorporation when the deficiencies were corrected by addition of vitamin B-12 and folic acid between 0 and 72 h in vitro. The mean percentages of the incorporation due to ATP-independent synthesis in nuclei from normal untreated cells, 5-fluorouracil-treated, cytosine arabinoside treated and vitamin B-12- or folate-deficient cells were 56 +/- 7% S.E., 41 +/- 7%, 84 +/- 3% and 28 +/- 6%, respectively. 5-Fluorouracil caused a two-fold increase in the cytoplasmic fraction of DNA polymerase when added to phytohaemagglutinin-stimulated lymphocytes between 48 and 72 h of culture but had no significant effect when added between 70 and 72 h.


Mitochondrial biogenesis in cultured mammalian cells. II. Mitochondrial protein and phospholipid synthesis in chloramphenicol-treated BHK-21 cells.

The effect of growth of BHK-21 cells in chloramphenicol on the synthesis of cellular proteins and phospholipids has been examined. The incorporation of leucine into total cellular proteins, or into the proteins of specific subcellular fractions are not significantly reduced by cell culture in the presence of chloramphenicol. In cells treated with cycloheximide, a small amount of chloramphenicol-sensitive labelling of protein was detected within the first hour of exposure to the drug. Chloramphenicol inhibits the incorporation of delta-amino-levulinic acid into hemoproteins, only if it is present during both the 48-h culturing and 4-h labelling period. De novo synthesis of cellular lipids as measured by pulse labelling with 32Pi or [3H]glycerol, is decreased in chloramphenicol-treated cells. This decrease is observed in all sub-cellular fractions, although the mitochondrial fraction is most affected. All phospholipids are affected, with diphosphatidylglycerol labelling reduced to the greatest extent. Although fatty acid synthesis is inhibited, the labelling of diphosphatidylglycerol with fatty acids is stimulated on chloramphenicol treatment.


Organization and transcriptional activity of brain chromatin subunits.

Digestion of brain nuclei with micrococcal nuclease produces 11.2-S chromatin subunits which comprise up to 80% of the total nuclear DNA. Hybridization studies with excess subunit DNA demonstrate that subunits are distributed throughout the repeated and nonrepeated sequences of the genome. No class of nonrepeated DNA appears to be excluded from subunits. Transcribed DNA is present in chromatin subunits since in vitro labelled poly(A+)-mRNA hybridizes to excess subunit DNA with kinetics identical to that for total DNA.


Action of heparin on mammalian nuclei. I. Differential extraction of histone H1 and cooperative removal of histones from chromatin.

Heparin interacts strongly with the histone component of chromatin, forming heparin-histone complexes which resist dissociation by 0.2 M H2SO4. Heparin treatment of unfractionated histones isolated from nuclei of Chinese hamster cells indicates that the affinities of the histone classes for heparin appear in the order from greatest to least: (H3, H4) greater than (H2A, H2B) greater than H1. However, when isolated nuclei are treated with heparin, H1 is released from the chromatin more readily than the other four histone classes. The release of these four histones (H2A, H2B, H3, and H4) is coordinate and occurs in a highly cooperative manner, as indicated by (1) dependence of the initial kinetics of histone removal upon heparin concentration, (2) analysis of DNA and histones in the fractions obtained from differential sedimentation of heparin-treated nuclei, and (3) analysis of the products from heparin-treated nuclei by equilibrium centrifugation in metrizamide density gradients. The results suggest rapid procedures for using heparin as an agent for studying the accessibility of histones in chromatin of intact nuclei. The relationship of these results to current models of chromatin structure is discussed.


Ontogenic characteristics of lactate dehydrogenase turnover in the mouse.

1. In order to investigate the ontogenic and turnover characteristics of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the foetal mouse, activities of this enzyme and isoenzyme distributions have been measured during development, and the time sequence of incorporation of radioactive amino acids have been determined during the last weeks of gestation. 2. In the early foetal stages, the enzyme was present with low specific activity and in the form of lactate dehydrogenase-5 in all regions, but a marked increase in specific activity became evident about the seventeenth day of gestation, and was accompanied by the formation of tissue specific patterns of isoenzyme distribution. 3. Measurement of turnover parameters was undertaken by both double-label ([3H] and [14C]leucine) and pulse-chase ([3H]leucine) techniques. Both procedures provided indications that appreciable degradation may occur during embryogenesis, with general proteolysis proceeding at a faster rate than with lactate dehydrogenase, and with higher rates of degradation occurring in foetuses which were smaller than average. 4. This data has been discussed in relation to the regional variation in these characteristics and the significance to studies of growth and differentiation.


Endopolygalacturonase from Rhizoctonia fragariae. Purification and characterization of two isoenzymes.

An electrophoretically homogeneous preparation of endo-polygalacturonase (poly(1,4-alpha-D-galacturonide)glycanohydrolase, EC 3.2.1.15) from culture filtrates of Rhizoctonia fragariae, a pathogenic agent in strawberry plants, was resolved into two isoenzymes when subjected to isoelectrofocusing in a narrow pH range. The isoelectric points of the two isoenzymes were 6.76 +/- 0.03 and 7.08 +/- 0.05. The two polygalacturonases exhibited similar substrate specificity, pH optimum and pattern of degradation of sodium polypectate. The two enzymes consisted of a single polypeptide chain which had an apparent molecular weight of 36 000 as determined by gel filtration on Sephadex G-100.


Purification of neuraminidase from influenza viruses by affinity chromatography.

The neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) of the influenza virus recombinant strain (HON2) was solubilized with detergents and isolated by affinity chromatography. The neuraminidase could be purified to a single high molecular weight glycoprotein when assayed under non-reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme showed an increase in specific activity from 2.46 to 189 micronM N-acetylneuraminic acid released per min per mg protein and the recovery represented 123% of the activity of intact virus particles. The enzyme could be purified from laboratory preparations of virus or from outdated influenza virus vaccine. Viral neuraminidases purified by this technique were stable at pH 6.0 for several hours.


The interaction of p-nitrophenyl carbamate with urease.

1. p-Nitrophenyl carbamate and thiourea have been shown to be substrates for urease (urea amidohydrolase, EC 3.5.1.5) 2. Urease has been shown to have a lower Km, 0.67 mM, with p-nitrophenyl carbamate than with urea, 2.0 mM. 3. The V of urease for the hydrolysis of urea, p-nitrophenyl carbamate and thiourea has been shown to be the same, indicating a common rate-limiting step. 4. A mechanism has been proposed for urease where the initial rate-limiting step is the release of a molecule of ammonia from the substrates.


Mechanistic studies on the biosynthesis of 6-ketoprostaglandin F1alpha.

The isolation, structure and biosynthesis of a novel metabolite of arachidonic acid formed during incubations of rat stomach homogenates is reported. This product was formed from unlabeled, tritium-labeled and deuterium-labeled arachidonic acid and the prostaglandin endoperoxides, G2 and H2, demonstrating its formation via the "prostaglandin endoperoxide synthetase" pathway. Its structure, 6-ketoprostaglandin F1alpha, was based on mass spectral interpretation of several derivatives (undeuterated and deuterated) and confirmed through chemical synthesis. 6-Ketoprostaglandin F1alpha is unique in being formed from a substrate of the "2" series of prostaglandins, i.e. arachidonic acid, yet belonging to the "1" series. A mechanism for its biosynthesis is proposed involving a specific cyclising enzyme which we term, 6(9)-oxycyclase.


The role of diacylglycerol-carrying lipoprotein I in lipid transport during insect vitellogenesis.

A diacylglycerol-carrying lipoprotein was isolated from mature eggs of the silkworm, Philosamia cynthia and compared, for physiochemical properties, with the major diacylglycerol-carrying lipoprotein I (LP-I) of hemolymph. The two molecules are identical an electrophoretic mobility, structural configuration as revealed by electron microscopy, and amino acid composition. In addition mannose was detected in the lipid-free protein moiety, thus enabling classification of the molecules as glycoproteins. The molecules differ in lipid content with egg-LP-I containing only 3.6% of the diacylglycerol content of hemolymph LP-I and the phospholipid and cholesterol components also showing a marked reduction. Analysis of the LP-I and vitellogenin (another diacylglycerol-carrying lipoprotein; LP-II) concentrations of mature eggs indicates that the amounts of the two glycolipoproteins in eggs are insufficient to account for the total acylglycerol content of the eggs. The results suggest that LP-I functions as a true carrier-protein serving to transport diacylglycerol from the fat body to the ovary. This proposal is supported by the observation that LP-I isolated from the egg retains the physiological capacity of hemolymph-LPi to take up diacylglycerol from fat body. Thus it is suggested that LP-I is the major source of lipid for vitellogenesis, whereas vitellogenin is the primary source of protein.


The primary structure of phospholipase A2 from porcine pancreas. A reinvestigation.

The primary structure of porcine pancreatic phospholipase A2 (EC 3.1.1.4) has been reinvestigated. A number of modifications have been introduced including the addition of a 7th disulfide bridge. The structure which is presented here shows a high degree of homology with the amino acid sequence of snake venom and horse pancreas phospholipase A2.


[Determination of the primary structure of alfalfa mosaic virus (strain S) coat protein. II. Complete sequence of the protein (author's transl)].

The alfalfa mosaic virus protein was submitted to the action of cyanogen bromide. Four peptides were isolated. Study of these peptides allowed us to determine the order. Then protein was submitted, after S-carboxymethylation or S-aminoethylation, to the action of different proteolytic enzymes: trypsin, chymotrypsin, thermolysin and papain. The peptides issued from these different hydrolysis were separated on Dowex 50 X4 and Dowex 1 X2, and their amino acid composition was determined. The use of classical methods of sequence determination, of mass spectrometry and for one case the use of a sequencer, lead to the obtention of the primary structure of all the tryptic peptides. The studies of chymotryptic, thermolytic and papainic hydrolysates, and of cyanogen bromide rupture, allowed us to isolate the overlapping peptides which were necessary for the reconstitution of the complete proteic chain.


Properties of carbohydrate-stripped thyroglobulin. III. Solubility characteristics of thyroglobulin.

In studying the solubility characteristics of thyroglobulin, it was found that the removal of sialic acid residues from either human or hog thyroglobulin by treatment with neuraminidase markedly decreased the solubility in salt solutions. On the other hand, naturally sialic acid-rich thyroglobulin obtained from human diffuse goiter was slightly more soluble than thyroglobulin in which sialic acid content was within the normal range. However, the desialized preparations prepared from both normal and sialic acid-rich thyroglobulin showed markedly lower solubility and further, showed no significant difference in solubility between them. These results indicate that the surface charge of thyroglobulin which influenced the salting-out property depends largely on the number of sialic acid residues in a thyroglobulin molecule, not on the degree of iodination of thyroglobulin.


Heterogeneity of purified cholera toxin.

The heterogeneity of Vibrio cholerae toxin, obtained from culture filtrates in homogeneous form by gel filtration and preparative disc gel electrophoresis has been studied. By means of disc electrophoresis on polyacrylamide gel cholera toxin was separated into three forms designated I (5%), II (15%) and III (80%). The toxic activity, amino acid content and molecular weight of the three forms were similar. The difference so far observed between the various electrophoretic fractions is a difference in net charge. Incubation of either cholera toxin II or cholera toxin III at relatively high pH leads to the formation of the more acidic forms. These forms, generated in vitro by deamidation of asparagine and/or glutamine residues, are indistinguishable from the toxins of similar electrophoretic mobilities isolated from crude culture filtrates.


Reactions of gold(III) ions with ribonuclease A and methionine derivatives in aqueous solution.

1. Addition of Au(III) (as chloroaurate) to folded ribonuclease A causes the formation of aggregates, as shown by chromatography and extreme broadening of the 270 MHZ 1H NMR spectrum. 2. When RNAase is partially unfolded Au(III) causes oxidation of methionines to the sulphoxide, and leads to almost complete unfolding (at molar equivalent ratios). 3. Reactions between model methionine derivatives and Au(III) show that the oxidation involves the production of Au(I)-methionine species. The stability of these complexes is dependent on the availability of free NH2 groups which catalyse their disproportionation.


Studies of disulfide bond location in ovine lutropin beta subunit.

By combinations of selective chemical cleavage (cyanogen bromide), selective enzymatic cleavage (trypsin, thermolysin), and random cleavage (partial acid hydrolysis) a series of disulfide-containing peptides have been isolated from ovine lutropin beta subunit. These peptides suggest six disulfide linkages between half-cystine residues in positions 23-72, 26-110, 93-100, 34-88, 9-90, and 38-57. The latter pair was placed by elimination of other possibilities. The first three pairs are in agreement with a report by Chung, D., Sairam, M. R. and Li, C. H. (1975) Int. J. Peptide Protein Res. 7, 487-493; the pair 93-100 has also been detected by Reeve, J. R., Cheng, K. W. and Pierce, J. G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155, using partial reduction and alkylation. In an attempt to improve the efficiency of enzymatic attack, a preliminary partial reduction as per Reeve et al. [16] was done. In this instance a peptide suggesting an additional disulfide linkage between half-cystines 23-26 was obtained as well as peptides consistent with the 23-72 and 26-110 placements. This was interpreted as an artifactual opening and recombining during partial reduction-reoxidation to produce the 23-26 linkage. The placement of three disulfide bonds (34-88, 9-90, and 38-57) is in disagreement with the pairings Chung et al. [15] suggest for these six half-cystine residues. Six reasons for uncertainty in the placement of disulfide bonds are discussed. It is concluded the definitive placement of the disputed three disulfide bonds in ovine lutropin beta subunit remains an open question.


Electronmicroscopic investigation of the flexibility of F-actin.

The contour lenghts and the end-to-end distances of a large number of F-actin filaments were measured in electronmicrographs. Preparation of F-actin for electron-microscopy was made at three different temperatures. The flexibility parameter or the elastic modulus for bending of F-actin was determined from the relation between the contour length and the end-to-end distance after statistical treatment. The value of the flexibility parameter obtained here showed good agreement with that obtained by the quasielastic light scattering at all temperatures examined. The flexibility of F-actin increased with raising temperature and with the binding of heavy meromyosin.


Isolation and characterization of a low-molecular weight seed protein from Ricinus communis.

A seed protein with an apparent molecular weight of 5000 has been isolated and purified from Ricinus communis. This protein has a very high content of glutamine and an unusual ultraviolet absorption spectrum. The amino-terminal sequence of 22 residues determined by automatic Edman degradation is: NH2-Pro-Ser-Gln-Gln-Gly-Cys-Cys-Gly-Gln-Ile-Gln-Glu-Gln-Gln-Asn-Leu-Arg-Gln-Cys-Gln-Glu-Tyr-.


Conformational transitions of alpha-elastin.

The alpha-elastin, the soluble product obtained from elastin by oxalic acid hydrolysis, has been studied from a conformational point of view in different conditions using circular dichroism spectroscopy. The spectral variations of the alpha-elastin dissolved in water and in different organic solvents/water mixtures have been examined in order to establish the effect of the solvents on the structural properties of the protein. The effect of calcium ions has also been studied on alpha-elastin dissolved either in water or in organic solvents. Finally, the effect of pH and temperature on the conformational properties of the alpha-elastin has been investigated.


Some observations on the ageing in vitro of reprecipitated collagen fibres.

The changes in solubility and amounts of reducible cross-links have been studied during "ageing" in vitro of reprecipitated rat skin collagen fibres by incubation at 37 degrees C. Fibres from pre-reduced collagen devoid of aldehyde precursors became insoluble at the same rate as that of normal fibres during "ageing". Insolubilization occurred at a much faster rate in the presence of oxygen than in air and was almost completely inhibited when oxygen was excluded. The rate of decline of the reducible cross-links was, however, unaffected by oxygen tension. The results indicate that, during "ageing" in vitro, conversion of the lysine-derived cross-links to a non-reducible form is not associated with solubility changes. The relationship of these in vitro changes to those ocurring in vivo is unknown.


Structural and functional studies of hemoglobin J Cala-bria: beta64 (E8) Gly leads to Asp.

A hemoglobin of high electrophoretic mobility was detected in a French male suffering from an acute leukemia; this hemoglobin was also present in his family. The variant was unstable and possessed an abnormal beta chain, in which a glycyl residue in position 64 (E8) was replaced by an aspartyl residue. This variant constitutes a new case of Hb J Calabria. Since the substituted E8 residue is in close spatial contact with that at B6, it was of interest to compare the properties of Hb J Calabria with those of other hemoglobins bearing substitutions at the same site.


Comparative effects of native insulin and insulin-dextran complexes on the metabolism of adipose tissue.

Insulin-dextran complexes have been prepared and their biological activities compared to that of native insulin. Complexes referred to as T-70 had a molecular weight of about 450 000, T-2000 being more than 2 million. Insulin-dextran T-70 was stable and no release of free insulin from the complex was observed upon its incubation with adipose tissue. Insulin-dextran T-70 mimicked the effects of native insulin upon adipose tissue metabolism. It also lowered blood sugar. These effects necessitated, on an insulin molar basis, concentrations of the complex that were 10-times greater than those needed for native insulin. Maximal concentrations of insulin complex T-70 or native insulin elicited similar quantitative effects. This suggested that when the concentration of insulin-dextran T-70 was high enough, the complex occupied a sufficient number of receptor sites to produce maximal stimulation of the tissue. In contrast, insulin-dextran T-2000 was barely effective, indicating that, probably due to its size, it was unable to reach receptor sites. The size, stability and metabolic effects of insulin-dextran T-70 observed in this study give additional support to the concept that insulin action is probably mediated via a series of events initiated at the level of the plasma membrane of adipocytes.


Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body.

Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous body by stepwise fractionation on AG-1X2(Cl-) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1:2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.


Dissociation of aggregated ferroheme complexes and protoporphyrin IX by water-soluble polymers.

The ferroheme-pyridine complex, ferroheme and protoporphyrin IX form the aggregates by the hydrophobic interaction in aqueous solutions. We found by spectrophotometric and fluorometric measurements that the aggregates dissociated into the monomers by the addition of water-soluble polymers, such as, poly(ethyleneoxide), poly(vinylalcohol), poly(vinylpyrrolidone) and poly(styrene sulfonate). The dissociation by the polymers proceeded as their hydrophobicities increased. The aggregated ferroheme was effectively dissociated by the copolymers of 4-vinylpyridine which were water-soluble polymer-ligands.


Studies on bacterial cell wall inhibitors. III. 3-amino-3-deoxy-D-glucose, an inhibitor of bacterial cell wall synthesis.

It was shown that 3-amino-3-deoxy-D-glucose, one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., inhibits the bacterial synthesis of cell wall. The antibiotic (100 microgram/ml) significantly inhibits the growth of Staphylococcus aureus FDA 209P as well as the incorporation of DL-[14C]alanine into the acid-insoluble macromolecular fraction of its growing cells in the presence of chloramphenicol (100 microgram/ml). In contrast, the antibiotic does not affect the incorporation of [3H]thymidine, [3H]uridine and L-[14C]leucine. The other constituents of kanamycin, 6-amino-6-deoxy-D-glucose and deoxystreptamine do not inhibit the synthesis of bacterial cell wall peptidoglycan.