Cyanide-insensitive oxidation of ascorbate + NNN'N'-tetramethyl-p-phenylenediamine mixture by mung-bean (Phaseolus aureus) mitochondria. An energy-linked function.

Freshly prepared washed or purified mung-bean (Phaseolus aureus) mitochondria utilize oxygen with ascorbate/tetramethyl-p-phenylenediamine mixture as electron donor in the presence of KCN. ATP control of the oxygen uptake can be observed with very fresh mitochondria. The electron flow, which is inhibited by antimycin A, salicylhydroxamic acid or octylguanidine, takes place by reversed electron transport through phosphorylation site II and thence to oxygen through the cyanide-insensitive pathway. Oligomycin and low concentrations of uncoupler partially inhibit the oxygen uptake in a manner similar to that observed for other energy-linked functions of plant mitochondria. An antimycin A-insensitive oxygen uptake occurs if high concentrations of uncoupler are used, indicating that the pathway of electron flow has been altered. The process of cyanide-insensitive ascorbate oxidation is self-starting, and, since it occurs in the presence of oligomycin, it is concluded that the reaction can be energized by a single energy-conservation site associated with the cyanide-insensitive oxidase pathway.


The effect of surgical trauma on muscle protein turnover in rats.

The rate of synthesis and catabolism of sarcoplasmic- and myofibrillar-muscle protein was measured in operated, sham-operated and food-restricted rats by using Na2 14CO3. The food-restricted group underwent sham operations and were limited to the food intake of the operated animals. Protein synthesis and catabolism were increased in the sarcoplasmic-muscle fraction in operated rats compared with that in sham-operated or food-restricted rats. The rate of synthesis of the myofibrillar protein decreased in operated animals, but the rate of catabolism was not altered in the myofibrillar-muscle fraction of the operated animals compared with that in food-restricted and sham-operated animals. In the operated animals, there was a net loss of protein from the muscle. Thus the rats that underwent surgery lost muscle protein, primarily as a result of a decrease in synthesis of myofibrillar protein. The changes in protein turnover in operated animals were not due to decreases in food intake, since protein turnover in sham-operated animals that were restricted to the food intake of the operated rats was not different from that in sham-operated rats fed ad libitum.


Studies on sex-organ development. Oestrogenic effect on ornithine decarboxylase activity in the differentiating Müllerian ducts and other organs of the chick embryo.

The activity of ornithine decarboxylase in the differentiating left and right Müllerian ducts was assayed and compared with that in other embryonic organs, i.e. the liver and the brain throughout the stages of development. In general the enzyme activity was high in the early stages and decreased extensively in the late stages of development. Specifically, in the left and righ Müllerian ducts, the enzyme activity was high from day 8 to day 9 of incubation. In the right duct the enzyme activity started to decline on day 9 and then continuously decreased to an almost undetectable value on day 18 of incubation. In the left duct the enzyme activity also decreased slightly from day 9 to day 12; however, it increased from day 13 to day 15 and finally decreased to a constant value from day 18 until hatching. The alteration in enzyme activity in the Müllerian duct as assayed in vitro during development is not due to the effect of the size of the endogenous ornithine pool. When the enzyme activity was subjected to oestrogen stimulation, an increase of 5--10-fold for the left duct and of 5--3-fold for the right duct was observed during the course of development. No such stimulation was observed with the treatment of progesterone. Testosterone consistently caused a 25--30% inhibition of the enzyme activity in the Müllerian duct. Oestrogen slightly stimulated the enzyme activity in the developing liver but inhibited that of the brain. The concentration of the three polyamines measured in the Müllerian duct corresponds to the activity of the enzyme determined.


Phospholipid composition and metabolism in mouse muscular dystrophy.

1. The composition and metabolism of phospholipids were studied in various tissues from both normal and dystrophic mice of the 129 ReJ strain. Phospholipids extracted from forebrain, spinal cord, sciatic nerve and plasma were fractionated by t.l.c. and measured. 2. Very significant alterations were found in the choline phospholipids from these tissues, except forebrain. Plasma phosphatidylcholine in the dystrophic mouse was increased by 38%. There was a 2-fold increase in lysophosphatidylcholine in the spinal cord of dystrophic mice. The sciatic nerve showed a marked decrease in sphingomyelin content, which is approximately half of that in the controls. 3. Five enzymes involved in phosphatidylcholine metabolism [namely cholinephosphotransferase (EC 2.7.8.2); phospholipases A (EC 3.1.1.4, EC 3.1.1.32); lysophospholipase (EC 3.1.1.5); lysophosphatidylcholine acyltransferase (EC 2.3.1.23); phospholipase C (EC 3.1.4.3)] were studied in tissue preparations from forebrain, spinal cord, sciatic nerves, gastrocnemius muscles and liver. 4. Activities of phospholipases A and C were significantly increased, about 5-fold and 60% respectively, in gastrocnemius muscle of dystrophic mice compared with controls. Phospholipases A also showed 50% higher activity in the sciatic nerves of dystrophic than of normal mice. Lysophosphatidylcholine acyltransferase activities were significantly increased in the sciatic nerves and spinal cord, by 50-100% over that of the controls. The forebrain and spinal cord from dystrophic mice, however, had only 60% of lysophospholipase activities of that of the normal control. Cholinephosphotransferase activity was unchanged in these tissues from both normal and dystrophic mice. 5. It is suggested that are number of features of mouse muscular dystrophy related to altered membrane structure and function can be rationalized in terms of changes in lipid composition and metabolism.


Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion.

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.


Membrane proteins in human erythrocytes during cell fusion induced by oleoylglycerol.

1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca(2+) used, at least 80% of the cells fused after 30min at 37 degrees C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in ;ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca(2+), although some cells fused when no exogenous Ca(2+) was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of ;ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1-2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca(2+) into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed.


Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes.

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.


Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.


alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.


The fate of 14C in glucose 6-phosphate synthesized from [1-14C]Ribose 5-phosphate by enzymes of rat liver.

1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1--30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3--17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the 'defined' pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation.


New reaction sequences for the non-oxidative pentose phosphate pathway.

1. Reactions leading to the formation of 14C-labelled volatile compounds and compounds volatile under acid conditions were investigated in a system actively synthesizing hexose 6-phosphates from [U-14C]ribose 5-phosphate by reactions catalysed by enzymes prepared from acetone-dried powder of rat liver; no reactions involving 14C-labelled volatile compounds were detected. Similarly the fixation of 14C-labelled volatile compounds into hexose 6-phosphate could not be detected. 2. A complete carbon balance was made for the reactants, intermediates and products of the reactions involved in the conversion of ribose 5-phosphate into hexose 6-phosphate by enzymes of rat liver. Five additional intermediates of pentose 5-phosphate metabolism in liver were detected, namely D-manno-heptulose 7-phosphate, D-altro-heptulose 1,7-bisphosphate, D-glycero-D-ido-octulose 1,8-bisphosphate, D-glycero-D-altro-octulose 1,8-bisphosphate and D-arabinose 5-phosphate. 3. D-Arabinose 5-phosphate was found to be utilized by a rat liver enzyme preparation to produce both hexose 6-phosphate and triose phosphate. 4. D-Arabinose 5-phosphate was reversibly converted into other pentose 5-phosphates. Paper chromatographic and enzymic evidence indicated that the conversion involved an enzyme tentatively named arabinose phosphate 2-epimerase, which catalyses the following reaction: D-arabinose 5-P in equilibrium D-ribose-5-P. 5. A variety of rat tissues also utilized D-arabinose 5-phosphate to produce both hexose 6-phosphate and triose phosphate and at a rate comparable with that obtained with D-ribose 5-phosphate. 6. A new reaction sequence for the non-oxidative pentose phosphate pathway in liver is proposed.


Stimulation of anterior pituitary prostaglandin E content and somatotropin (growth hormone) synthesis by phospholipase A.

The prostaglandin E content of dispersed rat anterior pituitary glands was found to increase in the presence of phospholipase A or arachidonic acid. The increases were abolished by the addition of indomethacin. Similarly, the rate of somatotropin (growth hormone) synthesis was increased by these two agents, and the increases were again abolished by indomethacin. Phospholipase A also stimulated somatotropin release. The stimulation of prostaglandin E accumulation was a specific response to those fatty acids that are precursors for prostaglandin synthesis. One such precursor, [3H]arachidonic acid, was incorporated by rat anterior pituitary glands in vitro, and found to be associated mainly with phosphatidylethanolamine-like material. It is concluded that the intracellular concentration of prostaglandin E is limited by the availability of precursor fatty acids and that this can be increased by the addition of exogenous precursors or by the action of exogenous phospholipase A on the cellular phospholipid. Factors that increased prostaglandin E concentrations also increase the rate of synthesis of somatotropin, providing further evidence for the concept that prostaglandin E is involved in modulation of the rate of synthesis of this hormone.


The mechanism of haem degradation in vitro. Kinetic evidence for the formation of a haem-oxygen complex.

The rate of haem degradation in aqueous pyridine shows a saturation dependence on O2 concentration. CO competitively inhibits the reaction. This is evidence for formation of an iron-O2 complex and is consistent with an intramolecular pathway for haem degradation.


Evidence for a reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed virgin and lactating rats.

Lipogenesis is increased in hepatocytes from fed lactating rats compared with virgin rats. Inhibition of lipogenesis with 5-(tetradecyloxy)-2-furoic acid resulted in increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased lipogenesis and esterification of [1--14C]oleate and decreased ketogenesis; these changes were reversed by the inhibitor. The reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed rats may be due to alterations in [malonyl-CoA] [McGarry, Mannaerts & Foster (1977) J. Clin. Invest. 60, 265--270; Cook, King & Veech (1978) J. Biol. Chem. 253, 2529--2531], but this mechanism is not considered to be sufficient to explain the increased ketogenesis in starvation completely.


Evidence for the participation of aspartate aminotransferase in hepatic glucose synthesis in the suckling newborn rat.

Inhibition of liver aspartate aminotransferase by L-2-amino-4-methoxy-trans-3-butenoic acid in the suckling newborn rat causes a decrease in all gluconeogenic precursors from phosphoenolpyruvate to glucose and an accumulation of lactate but not of pyruvate. This suggests that the aspartate shuttle is operative and confirms the quantitative importance of lactate as a gluconeogenic precursor at this time during development.


Evidence for a role of insulin in the regulation of lipogenesis in lactating rat mammary gland. Measurements of lipogenesis in vivo and plasma hormone concentrations in response to starvation and refeeding.

Fatty acid synthesis in the mammary gland of lactating rats in vivo was 5-fold higher than in the liver. Starvation decreased fatty acid synthesis in the gland 50-fold, whereas refeeding for 2h completely reversed this change. The plasma insulin concentration decreased 2-fold in starvation and was restored to the fed-rat value on refeeding. Glucagon and prolactin concentrations did not always change in parallel with lipogenesis, suggesting that insulin may be a regulator of this process in the gland.


A sub-population of rat liver membrane-bound ribosomes that are detached in vitro by carcinogens and centrifugation.

The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high  KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes.


Regression of naturally occurring atherosclerotic lesions in pigeon aorta by intestinal bypass surgery. Early changes in arterial cholesteryl ester metabolism.

Early changes in cholesteryl ester metabolism of the aorta during the regression of naturally occurring atherosclerotic lesions in pigeon aorta by ileal bypass surgery were examined. Three months after surgery, there was a decrease (50%) in the content of cholesteryl esters in the aorta. Increases in the activity of cholesteryl ester hypdrolase in the lysosomal (P less than 0.05) and the supernatant (P less than 0.01) fractions of the aorta also occurred at this time. There were no differences in the activity of cholesteryl ester synthetase and in the plasma levels of cholesterol and triglycerides between the ileal bypass group and the controls. These results suggest that ileal bypass surgery decreases the level of cholesteryl esters in the aorta, probably because of enhanced cholesteryl ester hydrolysis.


Blood lipids in Italy. Regional differences.

Serum cholesterol and triglyceride levels have been evaluated in samples from fasting males aged 20--59 in Northern (Brisighella), Central (Rome) and Southern (Pozzuoli) Italy. Regularly performed quality controls between laboratories assured comparability of data. A statisitically significant difference of mean serum cholesterol and triglyceride levels was observed for most age-groups in the 3 different areas, lower values being found in the southern population as compared to the central and northern ones. These results support previous findings and the thesis that large differences in blood lipid levels may still exist even within the same country and that they at least in part may be culturally determined in connection with different dietary habits.


Distinctive protein profiles obtained from extracts of normal and atherosclerotic human aorta.

Specific areas of fourteen autopsied abdominal aortas were layer-dissected, histologically graded and solubilzed with SDS, dilute saline or SDS-urea and beta-mercaptoethanol. Comparisons were made between intima, media, lesions of progressive severity and an in-vivo thrombus. Apparent molecular weights were calculated from the Coomassie blue stained gels of these extracts extracts and each band's contribution estimated by integrative densitometry. Four minor high moleucular weight bands and six medium to low molecular weight bands were detected in the arterial extracts. Band intensity stain patterns of normal intima are easily distinguished from normal media. Extracts of minor lesions resemble normal media; higher grade lesions demonstrate increased amounts of characteristic initmal bands. The major medial band is also seen on gels of thrombus extracts. Both of the bands most characteristic of atherosclerotic lesions stain for carbohydrate. Isolation and characterization of these (glyco)proteins will provide material for binding studies. Quantitation of characteristic lesion proteins may provide insights into the proliferative phase of this disease.


Platelet aggregation studies in coronary artery disease. Past 4. Effect of aspirin.

We evaluated platelet aggregation in vitro in blood samples drawn simultaneously from aorta and coronary sinus. Platelet aggregation was significantly lower in the coronary venous blood than in the aortic blood in patients with coronary artery disease. Lower platelet counts were also observed in coronary venous blood. No such differences were seen in subjects with normal coronary arteries. Oral administration of aspirin eliminated the differences in platelet aggregation and counts across the myocardial vascular bed. These observations suggest that platelet sequestration in the myocardial vasculature may be related to the presence of disease in the coronary arteries.


The relationship between serum cholesterol and fecal 7alpha-dehydroxylase activity in three ethnic groups in South Africa.

The ability of fecal bacteria to 7alpha-dehydroxylate primary bile acids was measured in vitro by incubating stool homogenates with labeled primary bile acids, and was compared to serum cholesterol levels in 4 South African groups: Rural Bantu (50 subjects), Urban White (20), Urban Bantu (17) and Urban Coloured (16). Mean serum cholesterol levels were 137 +/- 23, 213 +/- 51, 199 +/- 62 and 206 +/- 46 mg/100 ml, respectively. (Rural Bantu significantly different.) The in vitro conversion of cholic acid by stool homogenates of Rural Bantu was significantly slower than that of the Urban Whites or the Urban Bantu was significantly slower than that of the Urban Whites or the Urban Bantu in two-hour incubations. There was no difference of conversion rates in 24-hour incubations of cholic or chenodeoxycholic acids. Gas-liquid chromatographic analysis of stool homogenates showed little difference in the distribution pattern of fecal bile acids or neutral sterols. These data suggest that the activity of the intestinal bacterial flora to convert primary bile acids was significantly reduced in the Rural Bantu as compared to the other groups, corresponding with lower serum cholesterol levels. However, by the time the stools were excreted the degree of conversion was comparable in each group.


The effects of plasma exchange on cholesterol metabolism.

Four patients heterozygous for familial hypercholesterolaemia were treated by repeated plasma exchange with or without lipid-lowering drugs. Repeated plasma exchange without drug therapy in 3 patients was associated with a significant 18--28% decrement in plasma cholesterol level, comparing control with plateau values observed 3 weeks after exchange. Further decrements in plateau values followed the addition of lipid-lowering drugs used in combination, clofibrate--nicotinic acid or clofibrate--nicotinic acid--cholestyramine (range of total decrement 39--50%). Plasma exchange was associated with an increased excretion of endogenous faecal steroids, but this increase was completely abolished by the subsequent administration of clofibrate--nicotinic acid. This therapy prevented any increase in bile acid excretion with concomitant use of cholestyramine resin. Plasma exchange with drug therapy was associated with a sustained rise in plasma cholesterol specific radioactivity. In a fourth patient, clofibrate--nicotinic acid was administered prior to plasma exchange and led to a 24% fall in plasma cholesterol. Subsequent plasma exchange in this patient produced no sustained change in plasma cholesterol plateau level. In two patients, withdrawal of drugs allowed plasma cholesterol to return to pre-exchange control levels. These observations suggest that plasma exchange probably produced an increase in endogenous cholesterol synthesis and a mobilisation of tissue cholesterol. In relation to plateau cholesterol values 3 weeks after an exchange, the data suggested that the reduction in plasma cholesterol level with plasma exchange and drug therapy could have been achieved by intensive drug therapy alone.


Pyridinolcarbamate and experimental atherosclerosis. Correlation of hypocholesterolemic and antiatherogenic effects.

Rabbits were maintained for 12 weeks on either a control or hypercholesteremic dietary regime, or on comparable diets supplemented with pyridinolcarbamate (PDC) at a level of 30 mg/kg body weight/day. Blood was obtained from all rabbits prior to study and at two-week intervals for analysis of serum cholesterol, phospholipid phosphorus and triglycerides. Animals from each group were sacrificed at 4-week intervals for quantitative assessment of the degree of atherosclerotic involvement of the aorta. All animals in the four groups consumed their entire daily allowance (100 g) of their respective diets, and weight gains throughout the feeding period were comparable in the 4 groups. PDC given with the control chow diet had no effect on serum cholesterol levels but did result in persistent decrease in serum triglycerides and a variable decrease in serum phospholipids during the 12-week feeding period. None of the rabbits on the chow diet, with or without PDC, had any evidence of aortic lesions during the experimental period. Rabbits fed 1% cholesterol administered with chow exhibited markedly elevated levels of serum cholesterol and phospholipids, while serum triglycerides were not significantly different than in the control group. In these animals there was a rapid and progressive increase in aortic atherosclerosis throughout the study, and at 12 weeks plaque involvement was 74 +/- 8% of the aortic surface. Addition of PDC to the 1% cholesterol--chow diet resulted in significantly lowered levels of serum cholesterol and phospholipids, but these remained elevated compared to the control levels. There was also a dramatic reduction in the rate and extent of aortic plaque formation. Thus, after 12 weeks on diet, only 27 +/- 6% of the aortic surface showed evidence of atheroma. The data suggest that PDC significantly decreases the hypercholesteremia resulting from feeding 1% cholesterol to rabbits, and that this may be largely responsible for the antiatherogenic effect of this drug.


Studies of glycosaminoglycan composition and biologic activity of Vessel, a hypolipidemic agent.

Heparin-like glycosaminoglycans (GAG) were isolated from commerical Vessel and their biologic properties studied. Vessel was found to be a mixture of chondroitin sulfates, dermatan sulfate and heparin-like GAG. Chondroitin sulfates and dermatan sulfate in Vessel were hydrolyzed by chondroitinase ABC and the residual Vessel was fractionated on a Dowex-1 Cl- column eluting with a stepwise-increasing concentration of NaCl (1.2--4.0 M). The major fractions eluted at 1.6 M and 1.8 M NaCl were tentatively identified by chemical analysis as heparin-like GAG with somewhat lower sulfate content than standard heparin. Both fractions had lipoprotein lipase-releasing activity and anticoagulant activity similar to heparin, but 1.6 M NaCl fraction had a third of the anticoagulant activity of standard heparin. The 1.8 M NaCl fraction complexed with serum lipoproteins similarly to heparin. In preliminary studies cholesterol-fed rabbits treated with Vessel exhibited somewhat less atherosclerosis than controls.


Platelet factors and the human vascular wall. Part 2. Such factors are not required for endothelial cell proliferation and migration.

The effects of platelet factors on human umbilical venous endothelial cell DNA-synthesis at various serum concentrations were studied. While [3H]thymidine uptake of these cells was almost linearly related to the concentration of serum in the culture medium, no significant differences were found at any serum level between cultures treated with sera derived from either platelet-rich or platelet-poor plasma. The role of platelet factors in cellular migration into standardized mechanical "wounds" was also investigated following inhibition of the proliferative response by irradiation of endothelial monolayers. Repopulation of such "wounds" was not affected by platelet factors. We conclude that endothelial cells proliferate and migrate in vitro independently of platelet factors.


Effect of concentration of perfusing free fatty acid on arterial lipid synthesis in perfused normal and atherosclerotic rabbit aortas.

Normal and atherosclerotic rabbit aortas were perfused at physiological pressure for 1 hour with media containing various concentrations of [3H]oleic acid, between 0.5 and 2.0 mmoles/l, complexed to a fixed concentration 40 g/l of bovine serum albumin (BSA). The mass of free fatty acid (FFA), which entered the arterial wall and was subsequently utilised for lipid synthesis, was calculated from the measured specific activities of FFA in the perfusates. In normal tissue, at all concentrations of FFA in the perfusate, the highest rates of utilisation of perfusate FFA for arterial lipid synthesis were for phospholipids (PL) and triglycerides (TG), with only about 2% in cholesteryl esters (CE). In atherosclerotic tissue, at both low and high concentrations of perfusate FFA, about 25% of fatty acid entering arterial lipids was in CE. When the concentration of FFA in the perfusion medium was raised, the mass of FFA from the medium that was incorporated in the total arterial lipids, increased in both normal and atherosclerotic tissue. The increase was due in normal tissue, to significant increases in incorporation into FFA, lecithin (PC), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), TG and CE, whilst in atherosclerotic tissue it was due to increased incorporation into PC, PI, TG and CE. The results suggest that raised concentrations of FFA in blood may increase the rate of synthesis of lipids in normal and atherosclerotic tissue and thus exacerbate the accumulation of certain lipids such as cholesteryl esters, in fatty streak lesions of atherosclerosis.


Risk factors in pigeons genetically selected for increased atherosclerosis susceptibility.

A strain of genetically selected White Carneau pigeons (WC-2) with increased atherosclerosis at similar plasma cholesterol concentrations as randomly bred (RBWC) pigeons was studied to evaluate the commonly known risk factors for atherosclerosis. Indicators for the presence of hypertension, diabetes mellitus, "stress", hyperuricemia and hypothyroidism were determined. In pigeons fed the atherogenic diet, major differences in atherosclerosis were seen between WC-2 and RBWC. WC-2 pigeons had more aortic surface covered with plaque and greater concentrations of aortic nonesterified cholesterol, esterified cholesterol, uronic acid, and hydroxyproline, as well as a greater prevalence and severity of coronary artery atherosclerosis. For WC-2 and RBWC pigeons we found similar levels of hypercholesterolemia, mean blood pressure, plasma triglyceride and glucose concentrations. In addition, several other physiological variables such as plasma uric acid, calcium and phosphorus concentrations, adrenal and thyroid weights which have been implicated in the pathogenesis of atherosclerosis were similar. The findings indicate that the differences in extent and severity of atherosclerosis between WC-2 and RBWC cannot be explained by differences in the risk factors studied. Possible genetic regulation of atherosclerosis by mechanisms operable in the arterial wall of WC-2 pigeons is suggested.


Relationship between plasma concentration of luteinising hormone and intensity of lay in the domestic hen.

The concentration of plasma luteinising hormone (LH) in samples taken at frequent intervals from 6 weeks of age until the onset of lay, and also at 9 months after the onset of lay, was significantly greater in hens with a high rate of egg production than in comparatively poor layers. 2. The difference was most marked during the period 7 to 9 weeks of age when there was a transient increase in plasma LH concentration remained stable in comparatively poor layers. 3. The concentration of LH in plasma, particularly at 7 to 9 weeks of age, before gonadal growth, or during the prepubertal peak of LH secretion, at about 2 to 4 weeks before the onset of lay, may be a useful criterion in the selection of laying strains.


The metabolic effects of sodium dichloroacetate in the suckling newborn rat.

Subcutaneous injection of sodium dichloroacetate (1 microgram/ g body wt every 3 h) in suckling newborn rats caused in 6 h a fall of 2.5 mmol/l in blood glucose concentrations, and a rise of 2.4 mmol/l in total blood ketone body levels, but no change in the high levels of plasma non esterified fatty acids. Glucose utilization, measured after intraperitoneal injection of D-glucose (2 microgram/g body wt), was not increased in newborns injected with dichloroacetate. The hypoglycaemia resulted from a decrease in gluconeogenic rate, secondarily to a lowering effect of dichloroacetate on blood levels of lactate, pyruvate and alanine. The hypoglycaemia induced by dichloroacetate was completely reversed by injecting newborn rats with a mixture of gluconeogenic precursors (lactate, pyruvate and alanine). It is concluded that the high rate of gluconeogenesis observed in suckling newborn rats in sustained by an increased release of lactate and, to a much smaller extent of pyruvate and alanine, by peripheral tissues. This probably resulted from the low pyruvate dehydrogenase activity found in peripheral tissues of the newborn rat. The hyperketonaemia induced by dichloroacetate could result from an increased ketogenesis and/or a decreased ketone body utilization.


[Diagnosis, intensive therapy and surgery of peritonitis and enterocolic occlusions of thromboembolic origin].

The authors present a series of 14 cases of intestinal or colonic obstruction due to arterial ischema (7 cases) or mesenteric vein thrombosis (7 cases), for which they provide an analysis of pathogenesis, clinical aspects, surgical strategy, and intensive care. Mortality was 87.5% in cases of arterial origin and 42.8% in those of venous origin. The authors stress the need for diagnosing intestinal infarction right from the start, relying on clinical intuition and with the help of only a few clinical symptoms and objective signs. Surgically speaking, the best chance of survival is afforded by restoration of arterial or venous patency associated with whatever resection of the ileum or colon the anatomical situation may require.


Effects of different growth conditions on survival after irradiation in hypoxia of human cells (NHIK 3025) in vitro.

Cell cycle kinetics and radiation response under hypoxic conditions were analyzed with human cells of the line NHIK 3025. The cells were either kept in continuous exponential growth by frequent reculturing, or went through log and plateau phase for each passage (recultured weekly). The cell cycle time for weekly recultured populations in early log phase was shorter than for cells in continous exponential growth. Cells in continuous exponential growth were more sensitive to radiation than cells in log phase. The difference in sensitivity was not due to partial synchrony of weekly recultured populations.


[An approximate solution to the integral equation by using non serial cuts (author's transl)].

The problem formulated and resolved by WICKSELL is reformulated for a new technique of quantitative cytology utilizing non-serial cuts instead of serial sectioning. With a technique borrowed from  numerical analysis an approximate solution can be obtained. Theoretical results are in agreement with experimental results obtained using a mixture of red blood cells from humans and rats.


[Effect of amizil and arecoline on 45Ca uptake by rat brain nerve endings].

The effect of benactyzine (amizyl--the central cholinolytic) and arecoline (cholinomimetic) in vitro on the 45Ca uptake by the nerve endings (synaptosomes) of the rat brain was studied. Benactyzine proved to evoke a biphasic change of the isotope uptake, whereas arecoline induced an initial rapid increase of the 45Ca uptake. Benactyzine suppressed the influence of arecoline and of depolarization on the isotope uptake. A conclusion was drawn that an increase of the 45Ca uptake under the action of arecoline was connected with activization of the Na-K canals. On the contrary, benactyzine depressed the permeability of these canals for 45Ca and activated the calcium canals proper.


Further studies on the variables affecting rubella haemagglutination and haemagglutination-inhibition tests.

The different factors affecting rubella HA or HI were studied and the occurrence of rubella HA, also in absence of gelatine, albumin and Ca++, was shown. The influence on HA and HI titers of different systems--diluent and indicator erythrocytes--was also discussed.


HI rubella antibody determination. (A comparison of methods proposed for the removal of non-specific inhibitors).

The Authors consider the different methods so far proposed for the selective removal of non-specific inhibitors for HI rubella antibodies. They show that the best results are obtained by dextran sulphate and acetone treatments, while the kaolin and heparin-MnCl2 procedures should be avoided especially in the serological diagnosis of acute rubella infection. The presence of specific IgM was demonstrated by 2-mercaptoaethanol.


[Further observations on human infection by Listeria monocytogenes].

Two cases of isolation of Listeria monocytogenes type 1 from vaginal mucus of two pregnant women who underwent premature deliveries with death of foetuses are described.


[Atypical mycobacteria isolated from the water of the Simeto River at Catania].

From 25 samples of water taken in the course of 1975 from different points of the river Simeto have been isolated 11 strains of atypical mycobacteria, classifiable in Runyon's II, III and IV group. M. fortuitum has been most frequently found (6 strains), while M. gordonae, M. smegmatis, M. chelonei, M. engbaekii have been isolated only once. An unclassifiable strain belonging to Runyon's 3rd group has also been isolated. The importance of research of atypical mycobacteria in the environment from an epidemiological and epizootological point of view has been discussed.


[Immune response to toxoplasma infection].

The Authors studied the response of 49 subjects (23 affected by toxoplasmosis, 6 with systemic lymphopaties, and 20 apparently healthy controls) to the stimulation of lymphocytes both with an aspecific mitogen (PHA) and a specific one (toxoplasma antigen). No difference was found in the response to PHA, but when the specific antigen was used, patients with toxoplasmosis showed a significant increase of the lymphocytes stimulation index when compared to patients with systemic lymphopaties and to controls.


Cytotoxic assays of sera of tumor-bearing patients and inhibition of monocyte and granulocyte spreading activities.

Sera of patients with solid tumors showed cytotoxic antibodies against a selected panel of lymphocyte and granulocyte donors, typed for HLA antigens. The same sera were also studied for soluble circulating immune complexes and for the capability of inhibiting granulocyte and monocyte spreading. The percentage of rosette forming cells was also measured in the majority of patients. Some alterations of these immunologic parameters were significantly correlated with the presence of granulocytotoxic antibodies. These results suggested that some unknown factors in sera from patients with neoplasms may interfere with host-defence mechanisms, but the role played by cytotoxins needs further clarification.


Screening of HBsAg and post-transfusional hepatitis type B.

The Authors consider the different opinions expressed about the HBsAg screening in blood donors and its usefulness in preventing post transfusional hepatitis B (PTHB). They report data on the incidence of such a disease, as observed in their transfusional experience during the last 5 years, with a comparison between results obtained after CEP and RIA screening systems. The Authors point out the results of their retrospective investigation: the transfusion blood with a low HBsAg titre showed to be more infectious than the blood with high titre. Consequently, it is necessary to use more sensitive methods for HBsAg screening and possibly to increase their sensitivity or to combine them with other methods fit to evidence further markers of virus B.


[Role of chronic HBsAg carriers in intrafamilial diffusion of hepatitis B virus infection].

To assess the hepatitis B virus infection risk to household contacts exposed to HBsAg-positive chronic carriers, 169 family members of 20 healthy carriers of HBsAg and of 12 with chronic hepatitis were followed up prospectively for two to five years. All family members are investigated for the presence of serum HBsAg/antiHBs system and for the presence of liver disease. Our results indicate that the family contacts of HBsAg carriers with evidence of chronic liver disease are at greater risk of exposure to HBV than the contacts of healthy carriers and that acquisition of HBsAg is more common among males than among females. Evidence of exposure to HBsAg is very frequent in young age with a striking rise between 10 and 20 years. On the contrary acquisition of anti-HBsAg is more common after 40 years of age. These data suggest that persistent circulation of HBV in a family of chronic HBsAg carrier increases the risk of infection in household contacts and that HBV is eliminated in a more infective form from the carriers with chronic liver disease.


[HBsAg and anti-HBs in thalassemia non-transfused patients (and in their relatives)].

The sera obtained from 105 non transfused adult heterozygous thalassaemic subjects and from 119 members of 25 thalassaemic families have been tested with radioimmunoassay methods for the presence of HBsAg and anti-HBs. 11 out of 105 thalassaemic patients (10.4%) showed HBsAg in their serum and 41 (40%) anti-HBs antibodies. Family studies revealed that the incidence of HBsAg was 8.9% in 89 thalassaemic members and 10% in 30 non thalassaemic relatives. The incidence of anti-HBs was 34.8% and 13.3% respectively. The frequency of HBsAg in thalassaemic subjects, independently of blood transfusion as shown by our data, suggests some difficulty in eliminating the antigen. So these subjects become rather easily carriers of the antigen and source of the infection particularly in the family.


The problem of the identification and classification of staphylococci.

The Bergey's and ICSB schemes for the classification of staphylococci have been analyzed and in our opinion, it seems that we are still far from the final solution of the problem.


[Immunological examination in patients with brain tumor and the usefulness of PHA skin test (author's transl)].

The usefulness of in vivo skin test reaction to phytohemagglutinin to assess cell-mediated immunity was investigated in 109 subjects, including 37 normal controls, 35 patients without brain tumor and 37 patients with brain tumor. Intradermal injection at forearm of 10 microgram of Bacto-phytohemagglutinin-P (PHA-P) produced an erythema within 24 hours. Upon PHA skin reaction the mean diameter of erythema was 25.3 mm with the normal in the control and 18.6 mm with other diseases than brain tumor, while it was 13.0 mm with brain tumor. Furthermore when mean diameter of those with longer than 15.0 mm are classified as positive in the PHA skin test, the normal in the control revealed 89.2% positive and the patients with brain tumor indicated only 37.8% positive. A positive reaction was observed in 10 (77.0%) of 13 benign brain tumor and 5 (26.3%) of 19 malignant brain tumor. A correlation was found between in vivo and in vitro responses to PHA, and between PHA and PPD skin reaction. In repeated PHA skin test at intervals of about one month, the skin reaction to PHA of patients with brain tumor varied depending on the clinical states. Other advantages of this test are no presensitization and it can be used repeatedly. The PHA skin test is a useful and simple screening test for cellular immune function of patients with brain tumor and as a simple method applicable clinically indicating accurate immuno-reactivity the PHA skin tests have been carried out during the course of treatment with an interval of about one month repeatedly.


[Moyamoya phenomenon and Moyamoya diseases (author's transl)].

An unverified disease called "Moyamoya Disease" or "Spontaneous occlusion of the circle of Willis" has been recently reported as a disease entity by some Japanese researchers. Since the first report of this disease by Shimizu and the author in 1955, many cases have been reported not only in Japan but in many countries outside Japan. It has been already clarified either clinically or pathologically, that, in the Moyamoya Disease, the most important finding is the basal arterial occlusive change of unknown etiology and the Moyamoya Phenomenon is only nonspecific neuroradiological change as the extraordinary dilated collaterals via the striate arteries, perforators etc. However, the real cause of the arterial obstruction is still obscure in the so-called "true Moyamoya Disease". Further studies will be necessary in order to establish a new clinical entity related to the Moyamoya Disease. However, under existing situations, the Moyamoya Disease must be strictly differentiated from the Moyamoya Phenomenon which can be frequently observed among cases with basal occlusion of known and unknown origin.


[Immunological surveillance mechanism of a rat brain tumor model--study of time-lapse changes in the concomitant immunity of peripheral blood lymphocytes by microcytotoxicity assays (author's transl)].

A rat brain tumor model was prepared by semi-stereotactically implanting 1 x 10(6) RG--C6 tumor cells (chemically induced and maintained in vitro) in the right caudate nucleus of inbred WKA rat by a new method "Soft Agar Technique" which we developed. The tumor grew at a nearly fixed rate. We measured the maximum tumor area and the whole brain area of the rat brain which were sectioned coronary in series. We regarded the percentage of the tumor area to the whole area of the same section as tumor growth index. Microcytotoxicity assays of peripheral blood lymphocytes were performed at various stages of the tumor growth. RG--C6 cells were used as target cells, and they mixed with lymphocytes by Takasugi and Klein's method. Cytotoxicity index was higher in 22 cases (80.00 +/- 16.67; tumor growth index, 0--10) than in 28 cases of the control group (67. 25 +/- 19.68) and then gradually decreased with tumor growth (Fig. 2). At last cytotoxicity index went down to 41.55 +/- 24.94 in 11 cases (tumor growth index, over 31). These observations have a strong resemblance to other animal tumor models and suggest that the rat having brain tumor intra-axially have concomitant immunity especially in the first stage of tumor bearing.


[Anterior communicating artery aneurysms accompanied with anomalies or anterior communicating artery (author's transl) ].

The investigation was carried out on arterial anomalies complicated with 296 cases of single aneurysm of anterior communicating artery and on the etiological significance of the anomalies in the development of cerebral aneurysms. Either the fenestration in anterior communicating artery or the presence of more than 2 anterior communicating arteries was observed in 17 cases (5.7%). As the anomaly was duplicated in some of these cases, the total number of cases with anomalies of anterior communicating artery became to be 26 cases (8.8%) out of 296 cases investigated. This occurrence rate was not higher than those observed in other cerebral aneurysmal cases and the control cases, which were reported previously. There was no significant difference as to the occurrence rate of hypoplasia of the A1 portion between the cases whose aneurysms of anterior communicating artery were complicated with arterial anomalies and the cases without the arterial anomalies.


Adrenocortical suppression by synthetic corticosteroid drugs: a comparative study of prednisolone and betamethasone.

1 Single-dose studies were conducted to investigate the relative potency of prednisolone and betamethasone in suppressing adrenocortical function. 2 Betamethasone produced more profound suppression of plasma cortisol than an equivalent anti-inflammatory dose of prednisolone.


Pharmacokinetic and concentration-effect studies with intravenous metoclopramide.

1 Pharmacokinetic and concentration-effect studies have been carried out following intravenous injection of 10 mg metoclopramide hydrochloride to seven normal male volunteers. 2 It is proposed that a two-compartment model adequately describes the disposition of the drug which is rapidly distributed (T1/2alpha = 4.9 +/- 1.1 min) and eliminated (T1/2beta = 165.7 +/- 20.2 min). Total body plasma clearance of the drug is high (10.9 +/- 1.5 ml min-1 kg-1) and approximates to liver plasma flow. 3 Metoclopramide i.v. increases gastric emptying as measured by an ethanol absorption test (P less than 0.005). The duration of this effect is at least 3 h. 4 Ethanol given after i.v. metoclopramide administration produces significant sedation during the first hour and at 3 h (P less than 0.001). 5 The effect of metoclopramide on gastric emptying, and the degree of sedation induced by ethanol would appear to be related to plasma metoclopramide concentration. 6 Metoclopramide increases serum prolactin to 59 +/- 5.8 microgram/1 at 30 min after injection. There is a linear relationship (r = 0.809) between serum prolactin increase and plasma metoclopramide concentration.


Disopyramide serum and pharmacologic effect kinetics applied to the assessment of bioavailability.

1 Serum, urine and pharmacologic effect (prolongation of the QT interval) kinetics of the antiarrhythmic disopyramide have been investigated in eight volunteers after intravenous administration (2 mg/kg) and oral administration (300 mg) of the two commercially available preparations, Rythmodan (Roussel Laboratories) and Norpace (Searle Laboratories). 2 An open one compartment body model adequately described the kinetics of disopyramide in serum and urine. 3 After intravenous administration, the following average pharmacokinetic parameters were found: biological half-life, 7.8 h; total clearance, 95 ml/min; renal clearance, 54 ml/min; apparent volume of distribution, 60 litres. 4 After oral Rythmodan and Norpace, serum concentration profiles and urinary excretion data revealed significant differences in rates of absorption, times required to achieve peak serum concentrations and biological half-lives. These differences were largely due to the relatively slow absorption characteristics of Norpace. 5 The absence of hysteresis in plots of QT prolongation against disopyramide serum concentration after oral administration indicated that serum and pharmacologic effect kinetics were indistinguishable within a kinetically equivalent compartment. 6 Analysis of both serum and urine data showed that while Norpace had a significantly higher degree of bioavailability (P less than 0.005), the 5--15% difference between the two formulations should not normally be of any clinical significance.


Simultaneous analysis of dapsone and monoacetyldapsone employing high performance liquid chromatography: a rapid method for determination of acetylator phenotype.

1 A rapid, accurate and convenient technique for determination of acetylator phenotype of patients or subjects has not been available for routine clinical application. 2 An improved method for rapid and convenient determination of acetylator phenotype is described. 3 The plasma concentrations of dapsone (DDS) and monoacetyldapsone (MAD) were measured 3 h after a single oral 100 mg dose of dapsone using a specific and sensitive high performance liquid chromatographic assay. 4 The plasma concentration ratio of monoacetyldapsone to dapsone can accurately assess acetylator phenotype in patients or subjects. 5 The clinical applications for this method are discussed.


Comparison of the effect of drugs upon some commonly used measures of bowel transit time.

1 To study the effect of drugs on methods of assessment of gut transit time a double-blind study of the effect of single doses of placebo, oxyphenisatin, anthracene purgatives, (+)-norpseudoephedrine and a combined preparation of (+)-norpseudophedrine and senna was carried out in ten normal volunteers. Gut transit time was assessed by dye and radio-opaque marker methods. 2 It was possible to demonstrate the effect of the anthracenes but not oxyphenisatin on gut transit time. 3 More sophisticated statistical techniques were required to demonstrate the retarding effect of the sympathomimetic amine and its reversal by senna. 4 Statistical analysis shows that assessment of intestinal transit time by dye or pellet methods gives identical information.


Effect of surgery under general anaesthesia on antipyrine clearance.

1 The effect of surgery under general anaesthesia on drug metabolism was investigated in man. The activity of hepatic drug-metabolizing enzymes was evaluated by measurement of antipyrine clearance, before surgery and 3 days after surgery, in eighteen patients. 2 In those patients in whom the operation lasted 2 h or less, the postoperative clearance of antipyrine was significantly (P less than 0.05) increased by 48%; in those patients in whom the operation lasted 2--4 h, it was non-significantly decreased by 36%; in those patients in whom the operation lasted more than 4 h, it was significantly decreased by 47% (P less than 0.01). 3 It is concluded (a) that short operations are followed by increased activity of hepatic drug-metabolizing enzymes, whereas (b) protracted operations are followed by decreased activity. It is suggested (a) that the former effect may be a consequence of enzyme induction by drugs used for premedication and anaesthesia, whereas (b) the latter effect may be the result of major surgical trauma.


The effect on myocardial contractility of a new beta-adrenergic receptor blocking drug, penbutolol.

1 The inotropic effect of penbutolol, a new beta-adrenergic receptor blocking drug, has been compared with that of propranolol in two similar groups of six subjects each. 2 Inotropic changes were assessed from changes in myocardial contractility index [LV dp-dt/11 T] as well as the slope of the regression line relating LV dp/dt to LVED during incremental pacing. 3 Penbutolol was found to have a significant negative inotropic effect of an order similar to that of propranolol in the respective doses studied.


Evaluation of effect of acetylsalicylic acid using electrical stimulation on the forefinger of healthy volunteers.

1 In order to determine the optimal level of electrical stimulation, the degree of pain sensitivity produced by electrical stimulation on the volar side of terminal phalange of the right forefinger was studied in 22 male and female volunteers between the ages of 20 and 27 years. 2 A stimulation frequency of 400 Hz, width of 0.1 ms and electrical potential of 55 V was found to be optimal for study fo analgesics. 3 The effect of acetylsalicylic acid was studied in 40 healthy male volunteers between the ages of 20 and 25 years. Electrical stimulation was applied for 5 s under a single-blind method at a frequency of 400 Hz, width of 0.1 ms and electrical potential of 25, 35 and 55 V. Placebo, 250 mg, 500 mg and 1 000 mg of acetylsalicyclic acid were administered orally under double-blind method conditions. 4 As a result of this study, it was found that acetslsalicylic acid in a dose of 500 and 1 000 mg, was effective in relieving pain with statistically significant differences from a placebo.


The features of hepatic enzyme induction with glutethimide in man.

1 Sequential measurements of D-glucaric acid excretion were made in six healthy volunteers before, during and after 3 weeks' daily medication with glutethimide 500 mg. 2 There was a rapid rise in D-glucaric acid excretion within 2 days of starting medication and a rapid decline when it was stopped. 3 Antipyrine clearance and indocyanine green clearance were measured before and at the end of the 3 weeks' medication. 4 There was a 55% increase in antipyrine clearance but no change in indocyanine green clearance. 5 There was no correlation between antipyrine clearance and D-glucaric acid excretion. 6 Glutethimide causes rapid enzyme induction in man without concomitant rise in hepatic blood flow.


Age related differences in the disposition of acetanilide.

1 The metabolism of fifteen elderly hospital in-patients and fifteen young people was studied, using a gas chromatography mass spectrometer method. 2 The results suggest that there is no significant change in hepatic oxidation of acetanilide with age. 3 The concentrations of metabolites were however, significantly elevated in the older group. 4 These results illustrate the importance of the decline in renal function with age in the disposition of drugs.


Growth and transplantability of clonally related tumorigenic murine cell lines.

A malignant cell line derived from the s.c. inoculation of Adenovirus 12 into a CBA mouse has been isolated in vitro, cloned, and within 10 passages the clones have been investigated for their karyotype, morphology, growth rate, saturation density and response to plant lectin in vitro, and their tumorigenicity and growth rate in vivo. The cell lines rapidly acquired a highly heterogeneous karyotype, but remained homogeneous with respect to more complex physiological parameters. Examination of the cellular characteristics has indicated that the rate of growth of the cell lines in vivo, but not their tumorigenicity, may be related to their in vitro potentials. The clones responded differently to the cytotoxic effects of concanavalin A, but there was no correlation between the effect of the lectin and the malignant potential of the cells.


Thiophene analogues of the carcinogens benzidine and 4-aminobiphenyl: evaluation in vitro.

A biologically active molecule with one or more aromatic rings often retains its activity when one of these rings is replaced by an isosteric and/or isoelectronic aromatic ring. Consideration has been given to whether this effect can be expected to apply to aromatic organic carcinogens. The literature relevant to this topic has been reviewed and the thiophene analogues of the carcinogens benzidine and 4-aminobiphenyl have been synthesized and evaluated for potential carcinogenicity. The compounds prepared were 5-p-acetamidophenyl-2-thiophenamine hydrochloride (XIII), 5-phenyl-2-thiophenamine hydrochloride (XIV), N-(5-p-acetamido-phenylthiophen-2-yl)acetamide (XV) and N-(5-phenylthiophen-2-yl)-acetamide (XVI) (see Chart for structures). Each compound was evaluated in the Salmonella reverse-mutation assay of Ames and the cell-transformation assay of Styles. The activity profiles observed for these compounds in vitro were consistent with their known chemistry, and indicate potential carcinogenicity. However, their overall chemical and biological behaviour casts doubt upon whether they would be capable of eliciting tumours in vivo. Because it is important to establish the degree of reliance which can be placed upon in vitro predictions of potential carcinogenicity generated for structurally new compounds, one of the thiophene derivatives, N-(5-phenylthiophen-2-yl)acetamide ((XVI), is currently being evaluated for carcinogenicity in mice.


Influence of misonidazole on the incidence of radiation-induced intestinal tumours in mice.

C57BL mice were given local irradiation to 2 cm2 of the lower abdomen in the dose range 16--24 Gy. There were some early deaths, but mice dying between 50--240 days predominantly developed invasive adenocarcinomas of the intestine. When the radiosensitizer misonidazole was given in a single dose shortly before irradiation the proportion of mice developing tumours was higher, but the difference was not statistically significant. However, there was a significant increase in the incidence of multiple tumours, largely attributable to tumours arising in the rectum.


Enhancement by Wy-14,643, a hepatic peroxisome proliferator, of diethylnitrosamine-initiated hepatic tumorigenesis in the rat.

Diethylnitrosamine (DEN), at a concentration of 100 parts/10(6) in drinking water for 14 days, caused the development, by 48 weeks, of very few liver tumours in 5 of 18 (27%) male F=344 rats fed control diet. When the DEN treatment was followed one week later by continuous feeding of the hypolipidemic hepatic peroxisome proliferator, Wy-14,643, at 0.1% dietary level, all of 28 rats (100%) developed, between 38 and 48 weeks, a significantly higher number of liver tumours. Furthermore, laparotomy at 22 weeks revealed that several rats fed Wy-14,643 after DEN initiation had developed visible liver nodules, suggesting that Wy-14,643 also accelerates the appearance of these tumours. Administration of another peroxisome proliferator, clofibrate, at 0.5% level in the diet after DEN initiation, also caused a substantial enhancement of liver tumorigenesis. The enhancement of liver-tumour development by clofibrate, however, was less than that by Wy-14,643. The marked enhancing effect of Wy-14,643 may be due to its profound hepatomegalic and peroxisome proliferative properties.


Inter-site variation of oestrogen receptors in human breast cancers.

When large human breast cancers were assayed for oestrogen receptors at multiple sites, 5-fold differences were found in the numbers of oestrogen receptors, between the site within a tumour. This may result from variations in the cell:stroma ratio from site to site. Such differences could be significant when receptor levels in the tumour are low (less than 50 fmol oestradiol bound mg cytosol protein) since the classification distinction between hormone-sensitive and hormone-insensitive breast cancers is based upon numbers of oestrogen receptors detected by the assay. This problem might be remedied by assessment of the cell:stroma ratio in all assayed tumours, and by the combination of the cytoplasmic oestrogen-receptor assay with other hormone-receptor assays.


NMR in cancer, XIII: application of the NMR malignancy index to human mammary tumours.

One hundred and nineteen specimens of human mammary tissue taken from 112 individuals, were inspected by pulsed proton magnetic-resonance techniques (at 22.5 MH2). The purpose of the study was to evaluate the diagnostic capabilities of the nuclear magnetic resonance (NMR) technique with regard to the recognition of malignancy. The combination of two NMR parameters (spin lattice (T1) and spin-spin(T2) relaxation times) into a malignancy index produced better than 95% discrimination between the 2 populations of tissue on a case-by-case basis. The mean and standard deviations obtained were 2.002 +/- 0.351 for normal tissue, and 3.137 +/- 0.667 for malignant specimens. The probability that this difference is not significant is considerably less than 0.01. In addition, specimens of fibrocystic disease and fibrous mastopathy had indices of 2.263 +/- 0.503 and 2.151 +/- 0.505 respectively. Both groups yielded P values less than 0.01 when compared to the malignant specimens.


Repopulation of gamma-irradiated Lewis lung carcinoma by malignant cells and host macrophage progenitors.

Cellular repopulation in Lewis carcinoma irradiated with 60Co gamma-rays was examined by performing sequential cell-survival estimations using an in vitro soft-agar-colony assay. Following local irradiation (15--35 Gy) two distinct types of colony were seen: compact colonies with tightly packed cells and diffuse colonies with widely dispersed cells. Maximal diffuse colony formation in vitro was only obtained in the simultaneous presence of adequate numbers of compact colonies. After whole-body irradiation only compact colonies were observed. Only-cell survival data from compact colony counts correlated with cell survival estimated by the lung colony assay and we conclude that compact colonies are produced by clonogenic tumour cells. Cytochemical and immunological evidence showed that diffuse colonies were composed of macrophages. After local irradiation the initial kill of clonogenic tumour cells was dose dependent. At each dose level, repopulation began immediately and proceeded with a doubling time of about 1 day. Macrophage colony-forming cells (macrophage progenitors) per tumour were initially reduced by about 3 decades, but recovered very rapidly to reach pretreatment levels within 2 days. We conclude that at least two populations of clonogenic cells are present in Lewis lung carcinoma, tumour cells that repopulate irradiated tumours by in situ proliferation and host-macrophage progenitors that repopulate locally irradiated tumours by infiltration. The hazards of confusing host and tumour cell colonies in in vitro assay systems are stressed.


Cell yield and cell survival following chemotherapy of the B16 melanoma.

We describe in this paper cell survival studies, using in vitro clonogenic assays, performed on the B16 melanoma treated in situ with various cytotoxic agents. In addition we have determined the effects of these agents on the yield of cells obtained by trypsinization. In untreated tumours the mean cell yield was approximately 10(8)/g, which is 20--30% of the cells actually present in the tissue. The plating efficiency was approximately 40%. Most agents rapidly affected both cell yield and cell survival. For example, within 20--30 h, gamma-radiation and several alkylating agents reduced cell yield by about 40%. The cell yield change was associated with an increase in mean cell size. Cell yield was reduced even more (approximately 70%) by Vinca alkaloids. This large reduction was associated with extensive cell lysis, observed as an increase in the necrotic fraction of tumours from approximately 35% to approximately 70%. Adriamycin, bleomycin and Ara-C also produced a moderate reduction in cell yield (approximately 40%), but actinomycin D did not reduce cell yield and FU increased it by about 30%. Only gamma-radiation, cyclophosphamide, CCNU, BCNU and melphalan produced more than a 90% reduction in cell survival, although there was a small but measurable reduction with all other agents except vinblastine, HN2 and actinomycin D.


Influence of micrococcus, BCG and related polysaccharides on the proliferation of the L1210 leukaemia.

A comparative study of the effects of BCG, Micrococcus lysodeikticus, and a series of structurally related polysaccharides (complement triggers) on the non-specific and specific immune resistance against L1210 lymphoid leukaemia was carried out and commented on. In contrast with authors of earlier reports, we were unable to generate any effective non-specific or specific immunotherapy after the graft of 10(4) leukaemic cells to 8--10-week-old CDF1 mice. However, when mice were prevaccinated with irradiated (8 krad X-rays) cultured cells combined with 1 mg of bacterium or polysaccharide one month before grafting 10(4) cells, they were given an immunoprotection that was more pronounced with the i.p. than with the i.v. route. Prevaccinated mice were afforded a stronger immunoprotection when boosted repeatedly with 1mg injections of bacterium or polysaccharide after tumour challenge.


Treatment of childhood lymphocytic leukaemia with high white-cell counts.

Combination chemotherapy with cytosine arabinoside, cyclophosphamide and L-asparaginase (Asnase) was given to 22 children with acute lymphocytic leukaemia (ALL) with a white-cell count greater than 30 X 10(9)/1, and other features suggestive of poor prognosis. Complete remission was induced in all patients--in 19 after 2 courses of chemotherapy and in the remainder after a third course. During induction, neutropenia occurred in 18 and severe infection in 3. Anaphylaxis to Asnase occurred in 8 patients after the second course and one other had transient Asnase-induced diabetes. All patients received central-nervous-system prophylaxis after achieving remission, during which they were also treated with weekly vincristine and a 2-week course of prednisolone. Continuation therapy consisted of short cycles of intermittent chemotherapy and BCG inoculation or long cycles of intermittent chemotherapy +/- BCG. Life-table analysis shows 46% complete remission rate at 28 months, with 6 patients all in complete remission followed up between 28 and 41 months. There were minimal complications of continuation therapy, and BCG inoculation was well tolerated.


Survival and recurrences five years after selective treatment for breast carcinoma.

110 consecutively diagnosed breast-cancer patients in all stages were included in a study to evaluate a selective surgical and radiotherapeutical treatment. The surgical treatment was total mastectomy and exploration of the axilla, with lymphnode biopsy and peroperative cytological examination. Axillary dissection was done only when this examination showed metastases. No radiotherapy was given to the axilla in patients with lateral cancers in the absence of metastases, or with limited metastasization (no periglandular growth, no growth in apical nodes). In medial and central cancers, radiotherapy was applied to the parasternal and supraclavicular nodes irrespective of axillary involvement. A staging system with a combined clinical and histopathological classification was used and formed the basis for the selective treatment. The corrected 5-year survival for the whole material was 80%, for those without axillary metastasis (Stage I) 95% and for those with axillary metastasis (Stage II) 68%. Six women were alive with known distant metastases. Of 63 patients without identified axillary metastases at the time of surgery, axillary recurrences occurred in only 3 (5%). It was concluded that patients without axillary metastases can be reliably selected by the peroperative examination used, and that in this group simple mastectomy results in a high disease-free survival. Early diagnosis and a possible beneficial effect of the actual therapeutic programme might both have contributed to the high overall survival.


Pulmonary and pleural lesions in rheumatoid disease.

Lung and pleural involvement in rheumatoid disease is reviewed and discussed. Attention is drawn to the male preponderance, to the frequency with which lung infections occur in rheumatoid patients and to some problems posed in them by infection with mycobacteria. Pleural effusion, pulmonary nodules and fibrosing alveolitis are familiar in association with rheumatoid arthritis. Less familiar associations include upper lobe fibrosis, 'shrinking lung' and mixed pleural and pulmonary involvement. It is suggested that rheumatoid patients display a wider range of intrathoracic disease than seems generally appreciated.


Pulmonary necrobiotic nodules without rheumatoid arthritis.

Pulmonary necrobiotic nodules are the least common of the pulmonary lesions associated with rheumatoid arthritis. Histologically they are identical to subcutaneous rheumatoid nodules. Systemic rheumatoid arthritis usually precedes the development of pulmonary nodules. Seven cases where the pulmonary nodule appeared before or without the development of systemic rheumatoid arthritis are described. The typical histological and radiological features of necrobiotic nodules were found in each. In five of the seven the nodules were in more than one site and in five there were cavitating nodules. Spontaneous improvement occurred in one case. Pulmonary nodules preceded systemic rheumatoid arthritis in three cases and in the remaining four cases systemic rheumatoid arthritis has not yet appeared despite prolonged follow-up. In all patients, tests for rheumatoid factor have remained negative. The absence of circulating rheumatoid factor and systemic rheumatoid arthritis cannot exclude the diagnosis in these cases if the histological diagnosis is accepted as conclusive.


Pulmonary alveolar proteinosis: analysis of pulmonary washings.

Bronchopulmonary washings from a patient with pulmonary alveolar proteinosis who underwent therapeutic pulmonary lavage were examined. There were large amounts of protein and lipids of which main classes were phospholipid, cholesterol and free fatty acid. The major component of phospholipids recovered from the washings was lecithin rich in palmitic acid. We also found a high IgG level in the washings. Both the findings of free cholesterol in high concentration in the washings and the electrophoretic pattern of protein in the washings suggested that the alveoli-filling material of our patient was not wholely derived from passive transudation of plasma constituents.


Energy reserve and cation composition of irreversibly sickled cells in vivo.

Cation composition, cellular hydration, and adenosinetriphosphate (ATP) content were measured in irreversible sickle cells (ISC's) separated from the blood of patients with sickle cell anaemia. Total monovalent cation (Na+ + K+) content was markedly reduced in ISC's and this largely was due to cell K+ depletion. Corresponding to the reduced cation content, cells were dehydrated as indicated by a reduced mean cell volume. ISC's also appeared to be grossly depleted of ATP. These biochemical characteristics allow us to expand the definition of ISC's beyond morphologic characteristics. In addition, these chemical alterations provide a means for elucidating the mechanism of ISC production in vitro.


Erythrocyte calcium abnormalities and the clinical severity of sickling disorders.

We studied erythrocyte calcium levels and uptake in a group of patients with sickle haemoglobinopathies of different clinical severity in an attempt to relate these measurements to the production of irreversibly sickled cells and disease severity. Erythrocyte calcium levels were measured by atomic absorption spectroscopy and calcium uptake by isotopic means. In sickle cell anaemia, erythrocyte calcium content was elevated and the uptake of isotopic calcium increased under both oxygenated and deoxygenated conditions. There was a direct correlation between the numbers of irreversibly sickled cells and calcium uptake and an inverse relationship between calcium uptake and red cell potassium level. The clinical course of disease was milder in patients with high fetal haemoglobin levels, but there was no relationship between clinical course and calcium levels, calcium flux or irreversibly sickled cells. Our observations suggest that calcium accumulation and irreversibly sickled cell formation are related processes. The absence of good correlation between various biochemical and clinical parameters emphasizes the complexity of factors which modify the clinical course of this disorder.


Localization and role of calcium in the erythrocyte coat: effects of enzymes and storage.

The effects of various treatments on erythrocyte shape, surface, cell coat and calcium binding sites have been investigated by means of high voltage electron microscopy (HVM), scanning electron microscopy (SEM) and conventional electron microscopy (TEM). Papain caused the formation of small blisters within the cellular surface as well as crenation and 'budding' of the erythrocytes. After neuraminidase treatment, long filaments were observed to radiate from the surface of the erythrocyte. The other enzymes investigated, RNA'se DNA'se, phospholipase, protease and trypsin, produced no demonstrable effect on the cellular structure, nor (with the possible exception of trypsin) on the cell coat as seen by subsequent staining with ruthenium red. Putative calcium binding sites on and in the erythrocyte membrane were demonstrated. Following incubation with radioactive calcium, activity was found in the erythrocyte membranes. Calcium binding could be reduced by prior treatment of the erythrocyte with EDTA, neuraminidase, and to a lesser extent, by papain and trypsin. Other enzymes had no demonstrable effect. Stored erythrocytes showed a progressive diminution in calcium binding over a period of up to 4 weeks.


Hereditary pyruvate kinase deficiency: role of the abnormal enzyme in red cell pathophysiology.

Two new mutant Pks, electrophoretically identical but kinetically slightly different, are reported. These two clinically innocuous PK variants, encountered in two non-related subjects, have combined in their daughter to give a fully expressed haemolytic anaemia. The functional abnormalities of the daughter's PK (increased K0.5 PEP, abnormal response to FDP, increased urea and guanidine-HCl stability, abnormal isoelectrofocusing and electrophoretic patterns) were like those of the parents but more pronounced, except for thermostability that was normal in the proband although markedly decreased in both parents. The family examined demonstrates that there is no relationship between in vitro properties of the variant and the severity of haemolysis. The hypothesis is put forward that the cause of haemolysis in PK deficiency may be associated with another defect located in the red cell membrane.


Haem biosynthesis in rheumatoid disease.

The activities in blood of six enzymes of the haem biosynthetic pathway have been determined in 12 patients with rheumatoid disease, six of whom were anaemic. The porphyrin and porphyrin-precursor intermediary products of haem biosynthesis were also determined in blood, urine and faeces. No significant differences were found between anaemic and non-anaemic subjects. Failure of delta-amino-laevulinate synthase activity to increase in response to anaemia may be the nature of the marrow unresponsiveness suggested as one factor in the causation of the anaemia. Normal ferrochelatase activity and normal concentrations of free protoporphyrin support the view that iron is effectively unavailable although present in normal amounts. Coproporphyrinogen oxidase activity was significantly depressed.


Quantitative abnormality of an Aalpha chain molecular weight form in the fibrinogen of cirrhotic patients.

The molecular weight heterogeneity of fibrinogen from the whole plasma of 12 normal and seven cirrhotic individuals was examined by means of a novel two-dimensional sodium dodecyl suphate (SDS) gel electrophoretic technique. Fibrinogen was first separated from other plasma proteins on a large pore gel, cut out of the gel, reduced, and separated into its component Aalpha, Bbeta and gamma chains on a second gel. Fibrinogen was resolved into two major bands, I and II, on the first gel. The ratio of fibrinogen II to fibrinogens I plus II was approximately 0.3 (range 0.2-0.35) for both normals and cirrhotic patients. Two major molecular weight (mol wt) forms of Aalpha chain were present in normal fibrinogen I: Aalpha/I and/or Aalpha/2, mol wt 7 X 10(4) and 6.7 X 10(4) respectively. Normal fibrinogen II contained either one of these Aalpha chains plus one of the smaller Aalpha chains, Aalpha/6--10, accounting for the 3--4 X 10(4) mol wt difference between bands I and II. Aalpha/2 comprised 33% of the total Aalpha chains (range 27--41%) in normal fibrinogen I and approximately 25% of the Aalpha chains in normal fibrinogen II. In contrast, fibrinogen I from six out of the seven patients contained a lower percentage of Aalpha/2 (range 10--25%). Similarly fibrinogen II from these patients was deficient in Aalpha/2, although the protein contained normal levels of lower mol wt Aalpha derivatives. No correlation was found between per cent fibrinogen II and per cent Aalpha/2 in either normal or cirrhotic subjects. These results suggest that at least two independent processes are responsible for the observed levels of Aalpha heterogeneity in normals and cirrhotics and that the process controlling Aalpha/2 production is a abnormal in cirrhotic individuals. This decrease in Aalpha/2 does not affect the coagulability of fibrinogen. Fibrin monomer aggregation studies indicate that a serum component is, in part, responsible for the abnormally transparent clot formed from the plasma of cirrhotics.


Proposed structure for coenzyme F420 from Methanobacterium.

The low-potential electron carrier, coenzyme F420, was purified from Methanobacterium strain M.o.H. A yield of 160 mg/kg of wet-packed cells was obtained. Results of analysis of hydrolytic fragments and periodate oxidation products of the coenzyme, by infrared, UV-visible, 1H and 13CNMR spectrometry, mass spectrometry, and quantitative elemental analyses indicate that coenzyme F420 is: N-[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy-4-pentoxyhydroxyphosphinyl]-L-lactyl]-gamma-L-glutamyl]-L-glutamic acid. A convenient trivial name would be the N-(N-L-lactyl-gamma-L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate. Proof of structure by organic synthesis was not performed; the stereochemical configuration of the hydroxyl groups on the side chain as well as the position of the hydroxyl group on the aromatic ring require confirmation by organic synthesis of the molecule.


Mechanism of action of papain: aryldehydroalanines as spectroscopic probes of acyl enzyme formation.

The alpha,beta-unsaturated aromatic amino acids phenyldehydroalanine (PDA) and styryldehydroalanine (SDA) were synthesized and used as sensitive spectroscopic probes to study the acylation of papain by specific substrates and inhibitors. The spectral changes observed upon acylation of the enzyme with peptides containing these amino acids are large red shifts of the absorption maxima (lambda max) of the chromophores. The magnitudes of the absorption shifts were 49 nm (from 277 to 326 nm) for PDA peptide and 59 nm (from 318 to 377 nm) for SDA peptides. The following specific substrates were synthesized: Ac-Phe-Phe-PDA-OEt, Ac-Phe-PDA-NH2, Ala-Ala-Phe-SDA-OME, Ala-Ala-Phe-SDA-NH2, Lys-Ala-(o-benzyl)tyrosyl-SDA-OMe, and Lys-Ala-(o-benzyl)-tyrosyl-SDA-NH2. Similarly, the specific competitive inhibitors Ac-Phe-PDA (Ki = 5.3 X 10(-6) M), Z-Phe-SDA (Ki = 5.6 X 10(-5) M), Ala-Ala-Phe-SDA (Ki = 2.9 X 10(-5) M), and Lys-Ala-(o-benzyl)tyrosyl-SDA (Ki = 1.1 X 10(-5) M) were prepared. An additional chromophore was used to follow the noncovalent association of an inhibitor or substrate with papain, independently from the acylation or deacylation steps. This chromophore, which was introduced into the peptides at position P2, IS p-(p"-dimethylaminophenylazo) phenylalanine (DAP). The light absorption spectrum of DAP is dependent on its environment. The substrates Ala-Ala-DAP-SDA-OMe and Ala-Ala-DAP-SDA-NH2 and the competitive inhibitor Ala-Ala-DAP-SDA (Ki = 2.5 X 10(-6) M) were prepared. The noncovalent binding of these peptides to the active site of papain was followed either by the increase in the absorption at 480 nm or the decrease at 550 nm. With these petides the acylation and deacylation reactions could be followed simultaneously at 377 nm. The extent of acyl enzyme formation was found to decrease in a sigmoidal way with increasing pH, with a transition point around pH 5.5.


A vibrational analysis of rhodopsin and bacteriorhodopsin chromophore analogues: resonance Raman and infrared spectroscopy of chemically modified retinals and Schiff bases.

Resonance Raman spectroscopy has been used to study chemically modified retinal analogues involving chain substitutions, ring substitutions, or Schiff-base linkages. In addition, retinal fragments and fully deuterated retinals were investigated, and infrared spectra of the four isomers of retinal were obtained. Low-frequency resonance Raman spectra are also reported for all of the isomers of retinal, for the protonated and unprotonated Schiff bases of trans-retinal, for beta-ionone, and for trans-3-dehydroretinal. Band assignments were made to specific vibrational motions, and these assignments have led to a detailed understanding of the spectral features observed in the resonance raman spectra of the retinylidene chromophore in rhodopsin and bacteriorhodopsin.


Resonance Raman spectroscopy of squid and bovine visual pigments: the primary photochemistry in visual transduction.

Resonance Raman spectra of squid rhodopsin have been obtained under a variety of temperature and illumination conditions. The data have been characterized in terms of spectral contributions from squid rhodopsin, isorhodopsin, bathorhodopsin, lumirhodopsin, mesorhodopsin, P-465, and acid metarhodopsin. The results are compared with the spectral features obtained from bovine rhodopsin, isorhodopsin, and bathorhodopsin. The data support a proposed structure for the chromophore in bathorhodopsin which is not all trans, 11-cis, or 9-cis. This structure can be generated from either rhodopsin or isorhodopsin by a similar motion (simultaneously rotating chromophore carbon atoms 10 and 11 out-of-plane). Furthermore, we detect the same distinct bathorhodopsin vibrational modes when rhodopsin is illuminated between 4 and 100 K. This demonstrates that under steady-state illumination the light-induced chromophore structural alterations occurring at 4 K are very similar to those occurring at higher temperatures. Finally, our data indicate that bathorhodopsin is generated not only by structural transitions in the chromophore but also alterations in the opsin conformation as has recently been proposed[Lewis, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 549].


Resonance Raman spectroscopy of the retinylidene chromophore in bacteriorhodopsin (bR570), bR560, M421, and other intermediates: structural conclusions based on kinetics, analogues, models, and isotopically labeled membranes.

Resonance Raman spectra of various intermediates in the bacteriorhodopsin proton pumping cycle have been obtained at physiological and low temperatures. To interpret these data, spectra of modél compounds, bacteriorhodopsin analogues, and isotopically labeled membranes have been measured. These results demonstrate that a protein group interacts with the Schiff base proton and, thus, the chromophore in protonated bacteriorhodopsin species is not a simple protonated Schiff base. This accounts for the abnormally low frequency of the C=N+H vibrational mode in bacteriorhodopsin and other failures to model the chromophore in bR570 with a simple butylamine protonated Schiff base of all-trans-retinal. To obtain the resonance Raman spectrum of M412 at physiological pH and temperatures, a dual beam kinetic technique was developed. We demonstrate that in the fingerprint region of the resonance Raman spectrum M412 is modeled accurately by a simple unprotonated butylamine Schiff base of all-trans-retinal. Spectral resolution and the solution environment of the membrane suspensions play important roles in this conclusion. Kinetic resonance Raman techniques are also used to monitor the time evolution of the M412 species and the intermediates which precede it. We find spectral features in our kinetic data which can be assigned to L550, and we present evidence for a new unprotonated species (X) which occurs before M412. Single pass flow resonance Raman spectra of bR560 also have been obtained, and, although bR570 and M412 appear to have all-trans chromophores, there are 13-cis-like features in the spectra of bR560, L550, and X.


A circular dichroism study of the interaction of chlorpromazine with mouse brain tubulin.

A circular dichroism study with subsidiary ultracentrifuge measurements on the interaction of chlorpromazine with mouse brain tubulin establishes the previous inference from binding studies that the drug induces a change in the structure of the protein. Binding of the first mole of chlorpromazine causes an alteration in secondary structure, which is reversible with respect to drug concentration, without detected change in tertiary structure or significant change in the state of association of the protein. The conformationally altered tubulin binds additional chlorpromazine molecules without further change in secondary structure.


Platelet 5-hydroxytryptamine transport, an electroneutral mechanism coupled to potassium.

Transport of 5-hydroxytryptamine into plasma membrane vesicles isolated from porcine blood platelets is stimulated when a potassium gradient (in greater than out) is imposed across the vesicle membrane. This stimulation occurs in the absence of measurable electrical potential across the membrane. Addition of valinomycin induces a membrane potential of approximately 50 mV (interior negative) as estimated by uptake of the lipophilic cation triphenylmethylphosphonium, but has surprisingly little effect on 5-hydroxytryptamine transport. Addition of 2,4-dinitrophenol dissipates the valinomycin-induced membrane potential. In the absence of valinomycin, 2,4-dinitrophenol has no effect on 5-hydroxytryptamine transport but valinomycin and 2,4-dinitrophenol together inhibit transport, probably by dissipation of the K+ gradient. These results are consistent with an electroneutral mechanism in which 5-hydroxytryptamine influx is directly coupled to potassium ion efflux and argue against an electrogenic mechanism in which there is a net influx of positive charge with 5-hydroxytryptamine.


Primary structure of human erythrocyte glycophorin A. Isolation and characterization of peptides and complete amino acid sequence.

Peptides of glycophorin AMN were prepared by cyanogen bromide cleavage and by chymotryptic and tryptic digestion. Cyanogen bromide cleavage produces three fragments which account for the entire polypeptide chain. Trypsin and chymotrypsin cleave completely at several sites, but incompletely at sites within the glycosylated segment of the polypeptide chain. Some of the latter sites become accessible to proteolysis after desialation in addition to exposure of new sites for cleavage. The amino acid sequence of glycophorin AMN has been determined by manual Edman degradation, using both the direct Edman and the dansyl-Edman procedures simultaneously for determination of glycosylated amino acid residues. The automated procedure was used for sequence determination of a hydrophobic peptide. Glycophorin A is a polypeptide chain of 131 amino acid residues and contains 16 oligosaccharide units attached to the amino-terminal third of the molecule. Fifteen oligosaccharides are linked O-glycosidically to either threonine or serine residues and one complex oligosaccharide unit is attached N-glycosidically to an asparagine residue. Amino-terminal sequences are different for glycophorin AM and AN, the two forms of the glycophorin A molecule coded for by genes at the MN locus. The differences in sensitivity to proteases of various sites on glycophorin A seem to be due to heterogeneity in the carbohydrate components and not to differences in the primary structure of the polypeptide chains. This work contains a number of revisions and corrections of earlier preliminary reports [Segrest, J.P., Jackson, R. chem. Biophys. Res. Commun, 49, 964-969; Tomita, M., & Marchesi, V.T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964-2968].


Circular dichroism and magnetic circular dichroism of iron-sulfur proteins.

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for the 2-Fe ferredoxins from Pseudomonas putida and Spirulina maxima, Chromatium HIPIP, the 4-Fe ferredoxin from Bacillus stearothermophilus, and the 8-Fe ferredoxin from Clostridium pasteurianum. The spectral range spans the near-infrared, visible, and near ultraviolet. In all cases except oxidized 2-Fe ferredoxins, electronic absorption is observed continuously from less than 5000 cm-1 to above 30,000 cm-1. The CD spectra of the two 2-Fe ferredoxins are similar. In contrast, the CD of the 4-Fe and 8-Fe proteins, for a given 4-Fe cluster oxidation level, varies considerable with protein. MCD is less sensitive to protein environment than is CD. In the 2-Fe proteins, MCD at 5 T is appreciably smaller than the CD; in the 4-Fe and 8-Fe proteins, MCD and CD are comparable in magnitude. Both CD and MCD are more highly structured than the corresponding absorption spectra. The CD and MCD spectra reported provide a broader base than heretofore available for the characterization of iron-sulfur proteins containing 2-Fe and 4-Fe clusters and for the evaluation of electronic structural models for these clusters.


Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein.

Synthetic procedures have been developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2C1 and Pro-Phe-ArgCH2C1, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. For example, Pro-Phe-ArgCH2C1 inactivates plasma kallikrein 50% in 24 min at a concentration of 2 x 10(-8)M, while other trypsin-like proteases are less susceptible in inactivation than kallikrein, differing by a factor of 48 for plasmin and factors of 10(2)-10(5) for factor Xa, thrombin, and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2C1 (Ki = 0.078 micron) is about 60 times that for Ala-Phe-LysCH2C1(Ki = 4.9 micron), whereas human plasmin exhibits about the same affinity for the former affinity label (Ki = 1.3 micron) as for the latter (Ki = 0.83 micron). The rate constants for the irreversible step of the affinity labeling reaction, k2, are similar for affinity labels tested with the individual proteases: 0.35 min-1 for plasma kallikrein and 0.18 min-1 for plasmin.