UDP-glucosyltransferase activity of housefly microsomal fraction.

Housefly UDP-glucosyltransferase activity towards p-nitrophenol was demonstrated in a system in vitro. The activity is localized in the microsomal fraction, requires UDP-glucose, is slightly stimulated by Mg2+ and is activated optimally over a wide range of detergent concentration. Phenobarbital increases the enzyme(s) activity about 3-fold, with p-nitrophenol as substrate, which differs from the corresponding mammalian glucuronyltransferase.


Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate.

Soluble lactate dehydrogenase (EC 1.1.1.27) extracted from brain, skeletal and cardiac muscle and liver of rats, and purified isoenzymes LDH-1 and LDH-5, were incubated with sodium deoxycholate. Deoxycholate almost totally inactivated isoenzyme LDH-5 (A4), whereas it left isoenzyme LDH-1 (B4) unaffected. Tissue lactate dehydrogenase was inactivated to different degrees depending on the origin of the enzyme. Electrophoretic isoenzyme studies of tissue lactate dehydrogenase showed the loss of activity to be quantitatively related to the overall percentage of subunit A distributed among the homotetramer LDH-5 and the heterotetramers LDH-2, LDH-3 and LDH-4. It was concluded that subunit A of lactate dehydrogenase interacts selectively with deoxycholate, irrespective of its association with subunit B. Distinct changes in electrophoretic mobilities of deoxycholate-treated isoenzymes strongly indicated an indiscriminate binding of deoxycholate by all LDH isoenzymes, probably through hydrophobic interactions. The results suggest that the inactivation of the enzyme is non-competitive, but the basis of the selectivity of deoxycholate towards subunit A is not known at present.


Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens.

1. Human C1q, a subcomponent of the first component of complement, contains 67 disaccharides (glucosylgalactose) and 2.4 monosaccharides (galactose) linked to hydroxylysine in one molecule. It was found that 82.6% of the hydroxylsine residues were glycosylated. The suggestion of the possible existence of glucosylgalactosylhydroxylysine reported previously [Yonemasu, Stroud, Niedermeir & Butler (1971) Biochem. Biophys. Res. Commun. 43, 1388--1394] was confirmed. 2. The hydroxylysine-glycosides are not detected in the C-terminal, non-collagen-like, globular regions, but only in the collagen-like regions in the subcomponent C1q molecule. 3. Alpha 1(I) and alpha 2 in pig skin, alpha 1(II) in bovine cartilage and alpha 1(III) in bovine skin collagens contain 2.0, 2.2, 13.2 and 2.0 residues of hydroxylysine-glycosides per molecule, respectively. The percentage of hydroxylysine residues glycosylated in each of these chains is relatively low (on average 38%). 4. Neither the high percentage of hydroxylysine residues glycosylated nor the high values for the ratios of disaccharides to monosaccharides in the subcomponent C1q resembles that in alpha 1(I), alpha 2, alpha 1(II) and alpha 1(III). 5. Similarities between the extent of glycosylation of hydroxylysine residues in collagen-like regions in the subcomponent C1q molecule and that of the collagenous constituents of human glomerular basement membranes, aortic intima, skin A- and B-chains and of bovine anterior lens capsule are discussed.


A possible role for dehydrodihydroxylysinonorleucine in collagen fibre and bundle formation.

The concentrations of NaB3H4-reducible collagen cross-links were determined at the time when collagen fibres and bundles are observed in electron micrographs of connective tissue developing around the implanted Ivalon sponge in adult male rats. The highest radioactivity occurs with hydroxylysinonoreleucine and histidinohydroxymerodesmosine, and the lowest with lysinonorleucine, the reducible amounts of these cross-links remaining relatively constant as fibres and bundles appear. On the other hand, dihydroxylysinonorleucine amounts are low during the initial stages of connective-tissue formation and rise sharply as collagen fibres and bundles develop and collagen matures, as shown by increased resistance of insoluble collagen to digestion with bacterial collagenase. The bulk of hydroxylysinonorleucine and dihydroxylysinonorleucine is glycosylated, the former with galactosyl or glucosylgalactosyl residues and the latter with glucosylgalactosyl residues. The changing relationships between the amounts of 3H-labelled hydroxylysinonorleucine, glucosylgalactosyldihydroxylysinonorleucine and non-glycosylated dihydroxylysinonorleucine as fibres and bundles appear suggest three post-translational steps involving lysyl-derived cross-links in the organization of collagen into fibres and bundles.


Multiple inhibition of glutathione S-transferase A from rat liver by glutathione derivatives: kinetic analysis supporting a steady-state random sequential mechanism.

Glutathione derivatives inhibit glutathione S-transferase A [cf. Biochem. J. (1975) 147, 513--522]. The steady-state kinetics of this inhibition have been investigated in detail by using S-octyglutathione, glutathione disulphide and S-(2-chloro-4-nitrophenyl)glutathione: the last compound is a product of the enzyme-catalused reaction. Interpreted in terms of generalized denotations of inhibition patterns, the compounds were found to be competitive with the substrate glutathione. Double-inhibition experiments involving simultaneous use of two inhibitors indicated exclusive binding of the inhibitors to the enzyme. The discrimination between alternative rate equations has been based on the results of weighted non-linear regression analysis. The experimental error was determined by replicate measurements and was found to increase with velocity. The established error structure was used as a basis for weighting in the regression and to construct confidence levels for the judgement of goodness-of-fit of rate equations fitted to experimental data. The results obtained support a steady-state random model for the mechanism of action of glutathione S-transferase A and exclude a number of simple kinetic models.


Comparison of endogenous and exogenous RNA primers of poly(U) polymerase in rat hepatic ribosomes.

The present work indicates that RNA primer requirements for poly(U) polymerase in the free ribosomes of the rat liver depend upon the degree of enzyme purification. The poly(U) polymerase activity obtained from a crude free ribosomal preparation was compared with the enzymic activity of a partially purified enzyme. After preliminary purification, the enzyme was fractionated by chromatography on Sephadex G-150 and CM-cellulose. Our results demonstrate the presence of several forms of poly(U) polymerase activities, some requiring exogenous RNA and others possessing their own endogenous primer RNA.


Effects of nucleophiles on the breakdown of the benzylpenicilloyl-enzyme complex EI formed between benzylpenicillin and the exocellular DD-carboxypeptidase--transpeptiase of Streptomyces strain R61.

Serine is one of the enzyme residues with which benzylpenicillin collides as a result of its binding to the Streptomyces strain-R61 DD-carboxypeptidase-transpeptidase enzyme. Nucleophilic attack occurs on C(7) of the bound antibiotic molecule with formation of a benzylpenicilloyl-serine ester linkage, i.e. formation of the benzylpenicilloyl-enzyme EI complex. To reject the bound penicilloyl moiety and consequently to recover its initial activities, the strain-R61 enzyme has developed two possible mechanisms. Pathway A is a direct attack of the serine ester linkage by an exogenous nucleophile, resulting in the transfer of the benzylpenicilloyl moiety to this nucleophile. In pathway B, the benzylpenicilloyl moiety is first fragmented by C(5)-C(6) cleavage and the enzyme-bound phenylacetylglycyl residue thus produced is in turn transferred to the nucleophile. Pathway B occurs with water, glycylglycine and other amino compounds. Both pathways A and B occur with glycerol, other ROH nucleophiles and neutral hydroxylamine. The nucleophilic attacks are enzyme-catalysed.


Liver monoamine oxidase in the obese-hyperglycaemic (ob/ob) mouse.

1. The specific activity of monoamine oxidase was found to be greater in liver mitochondria from ob/ob mice than from lean mice. The activities of marker enzymes were similar in both tissues. 2. Experiments with various substrates (5-hydroxytryptamine, benzylamine and tyramine) and inhibitors (clorgyline and deprenyl) indicated that, unlike rat liver mitochondria, mouse liver mitochondria contain a predominance of the B-form of monoamine oxidase. 3. The Km values for lean and ob/ob mice were the same for any given substrate and were in the increasing order 5-hydroxytryptamine less than tyramine less than benzylamine. Vmax. was approximately 50% greater in obese than in lean mice. 4. Extraction of liver mitochondria with acetone/water or acetone/water/NH3 to remove lipids decreased the enzyme activity relatively more in obese- than in lean-mice preparations, but residual activity was the same in both preparations.


Persistence of methylated bases in ribonucleic acid of syrian golden hamster liver after administration of dimethylnitrosamine.

1. Syrian golden hamster liver ribosomal RNA was isolated up to 96 h after administration of [14C]dimethylnitrosamine at 25 mg/kg or 2.5 mg/kg body weight. 2. The chemical alkyation products, 7-methylguanine, 3-methylcytosine, O6-methylguanosine and 1-methyladenosine, were measured after acidic or enzymic hydrolysis of the RNA to bases or mononucleosides followed by ion-exchange chromatography. 3. Between 7 and 96 h, the relative amounts of alkylation products did not change with time even though the absolute amounts fell by approx. 80% and 51% after the high and low doses respectively. 4. The results suggest that base specific excision repair does not exist for RNA alkylation products in this experimental system.


A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations.

By using the fluorescence enhancement of ethidium bromide when it binds to RNA, a very rapid, simple and sensitive assay for the concentration of ribosomal RNA in complex mixtures has been devised.


Cholesterol exchange as a function of cholesterol/phospholipid mole ratios.

The activation energy (Ea) for cholesterol exchange between dioleoyl phosphatidylcholine vesicles and erythrocyte 'ghosts' is measured as a function of molar percentage of cholesterol in both donor and acceptor membranes. A sharp increase in Ea occurs (from 39.9kJ/mol to 84kJ/mol) when the molar percentage of cholesterol decreases from 30 to 20%.


S-(N-dansylaminoethyl)-6-mercaptoguanosine as a fluorescent probe for the uridine transport system in human erythrocytes.

A fluorescent derivative of 6-mercaptoguanosine, S-(N-dansylaminoethyl)-6-mercaptoguanosine, was synthesized, and found to be a strong inhibitor of the uridine transport system of erythrocyte (Ki approximately 0.3 microM). The emission spectrum of this compound has peaks at 400 and 550 nm. The emission at 550, but not that a 400 nm, in environment-sensitive. A method was devised for preparing a suspension of erythrocyte-membrane fragments with sufficiently low light scattering so that a detailed study could be made of the fluorescence of the probe when bound to membranes. Direct binding measurements showed the existence of a tight binding site, with a dissociation constant of the same order of magnitude as the inhibition constant. Binding of probe and substrate are not mutually exclusive, but the fluorescence and affinity of the bound probe are sensitive to the presence of uridine. The emission spectrum suggests that the bound probe penetrates into the bilayer region of the membrane.


Sodium ion-coupled uptake of taurocholate by intestinal brush-border membrane vesicles.

The uptake of taurocholate was studied in membrane vesicles isolated from brush borders of hamster jejunum and ileum. When an extra- to intra-vesicular gradient of Na+ ions was present ileal vesicles took up 10 times more taurocholate than did jejunal vesicles. Accumulation of taurocholate by ileal vesicles was transient and was due to transport of this bile salt into an osmotically active intravesicular space rather than simple binding. Uptake of taurocholate was specifically dependent on Na+ ions; NaCl and Na2SO4 were capable of supporting accumulation, whereas KCl, LiCl and mannitol were not. Na+-coupled uptake of taurocholate into ileal vesicles was inhibited by other trihydroxy bile salts, by preloading the vesicles with Na+ and by simultaneous flow of glucose into the vesicles. Similarly, vesicular uptake of glucose was inhibited by simultaneous uptake of taurocholate. These results demonstrated that brush-border membrane vesicles prepared from ileum possess an Na+-coupled co-transport system for taurocholate that is similar to the active bile-salt transport system present in the intact ileum.


Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride.

1. The microsomal haem oxygenase activity induced by the administration of CoCl2 was found mainly in the smooth-surfaced microsomal fraction, whereas that of the untreated control animals was widely distributed in smooth-surfaced microsomal, rough-surfaced microsomal and Golgi fractions. 2. When microsomal preparation was incubated and the time course of the distribution of biliverdin between the membranes and the medium was followed, most of the biliverdin formed was found first in the medium. This suggests that the active site of haem oxygenase is exposed on the cytoplasmic surface of the membranes. The possible localization of the enzyme at the outer surface of the membranes was also supported by a digestion experiment with trypsin. The haem oxygenase activity was greatly decreased even at low concentration of the proteinase, which did not affected the NADPH-cytochrome c reductase activity. 3. When microsomal preparation was further fractionated by isopycnic centrifugation in the presence of deoxycholate or by partitioning of sonicated microsomal preparation in aqueous-polymer two-phase systems, most of the haem oxygenase activity was found in a fraction different from the main fraction of the NADH- and NADPH-cytochrome c reductase and NADH--ferricyanide reductase activities. This indicates the different distribution of haem oxygenase from the other enzymes mentioned, on the lateral plane of microsomal membranes, and suggests the different localization of the haem oxygenase system from the electron-transport system linked with cytochrome b5 and cytochrome P-450.


The effect of starvation on the rate of protein synthesis in rat liver and small intestine.

1. A method is described that allows for measurement of protein synthesis in liver and intestine in the rat. By injecting a massive amount of [14C]leucine (100 mumol/100 g body wt.) an attempt has been made to over come problems of precursor specific radioactivity and problems arising from the breakdown of labelled protein that are encountered when tracer amounts of amino acids are used. 2. Starvation for 2 days resulted in decline in the rate of total liver protein synthesis from 87%/day to 62%/day. 3. In jejunal mucosa the rate of protein synthesis was 136%/day. This declined to 105%/day after 2 days of starvation.


Polyamine replacement by magnesium ions in BHK-21/C13 cells.

Cultures of BHK-21/C13 cells, whose growth was inhibited by deprivation of serum, were stimulated to grow by addition of serum to the culture medium. Addition of MgCl(2) to the medium, to increase the concentration of Mg(2+) ions by 15mm, 30min before addition of serum, had no effect on the stimulation of cell growth, but inhibited the accumulation of cellular spermidine, so that the spermidine/spermine molar ratio was lower in these cultures than in cultures that had received no additional cations. The increase in the activity of ornithine decarboxylase that occurs 4-5h after serum ;step-up' was substantially diminished by increasing the concentration of Mg(2+) ions, but not of Na(+) or K(+) ions, in the medium by 30mm, 30min before addition of serum, and this inhibition was maintained for at least 24h. Methylglyoxal bis(guanylhydrazone), added to serum-deprived cultures to a concentration of 20mum, 30min before addition of serum, severely inhibited the increase in cell growth. The inhibitory effects of the drug were prevented by simultaneous addition of spermidine to the medium (to 100mum), and were partly prevented by the simultaneous addition of Mg(2+) ions (to 30mm). Mg(2+) ions were particularly effective in overcoming the inhibitory effect of methylglyoxal bis(guanylhydrazone) on the synthesis of DNA. Thus although a certain lack of specificity for cations exists in BHK-21/C13 cells, in that Mg(2+) ions can be substituted for polyamines, particularly spermidine, to some extent, there are cellular processes for which the requirement for polyamines as cations is specific.


Inhibition of the discharge of endocytosed protein from phagosomes into lysosomes in hepatoma cells exposed to dimerized ribonuclease A.

The mechanism of the cytostatic action of dimerized ribonuclease A toward cultured hepatoma cells was investigated. A decrease in mitotic index, modifications of adsorptive properties of the pericellular membrane and inhibition of the degradation of two different proteins taken up by endocytosis are the first cell functions to be affected by the dimer. This effect on protein digestion is not due to an inhibition of proteolytic enzymes. The intracellular localization of exogenous protein and of ribonuclease dimer was studied by cell fractionation. When proteins (horseradish peroxidase or rabbit immunoglobulin G) are taken up by control hepatoma cells, they are first associated with phagosomes equilibrating at a lower density than lysosomes; their density distribution gradually becomes similar to that of lysosomes. When cells are pre-exposed to ribonuclease dimer, this modification of the density distribution as a function of time no longer occurs, although these proteins are still intracellular, as indicated by fractionation by differential centrifugation. During the first hour after addition of ribonuclease dimer, kinetic studies show an increased fixation of peroxidase to the cell membrane. Protein release into the culture medium is also increased. These results can be explained either by an absence of fusion between phagosomes and lysosomes, or by an inhibition of the discharge of peroxidase adsorbed to the phagosomal membrane after fusion.


The effect of colchicine on human blood platelets under conditions of short-term incubation.

The effects of colchicine on ADP-induced aggregation and on the phosphorylation of tubulin-like protein from human blood platelets were studied. Colchicine at 2mM concentration completely inhibits ADP-induced aggregation after 8min incubation. Under the same inhibitory conditions, phosphorylation of tubulin-like materials in intact platelets was also impaired whereas the endogenous kinase activity of tubulin, isolated through polymerization--depolymerization cycles, was not affected. It was also shown that, under conditions of maximal inhibition of both aggregation and tubulin phosphorylation, colchicine does not penetrate into the cells. The results obtained suggest that the effect of colchicine on platelet aggregation might be mainly, although not exclusively, due to a non-specific effect of the alkaloid on the plasma membrane, rather than to a direct action of the drug on the microtubular protein subunits.


The influence of vasopressin and related peptides on glycogen phosphorylase activity and phosphatidylinositol metabolism in hepatocytes.

The relative abilities of seven vasopressin-like peptides to activate hepatic glycogen phosphorylase and stimulate phosphate incorporation into phosphatidylinositol were compared. Although the individual peptides differed in their potencies, the concentrations required to stimulate phosphatidylinositol metabolism were always greater (about 10 times) than those needed to activate phosphorylase. The molecular specificity of the hepatic vasopressin receptor and the role of vasopressin-stimulated phosphatidylinositol turnover are discussed.


Leucine degradation in cell-free extracts of skeletal muscle.

Since skeletal muscle is the major site in the body for oxidation of leucine, isoleucine and valine, the pathway and control of leucine oxidation were investigated in cell-free preparations of rat muscle. Leucine was found to be transaminated to 4-methyl-2-oxopentanoate, which was then oxidatively decarboxylated. On differential centrifugation 70--80% of the transaminase activity was recovered in the soluble fraction of the cell, and the remaining amount in the mitochondrial fraction. The transaminase, from both fractions had similar pH optima and both were markedly inhibited by Ca2+. Thus changes in cellular Ca2+ concentration may regulate transaminase activity. Both transaminases had a much higher affinity for 2-oxoglutarate than for pyruvate. Therefore the utilization of amino groups from leucine for the biosynthesis of alanine in muscle [Odessey, Khairallah & Goldberg (1974) J. Biol. Chem. 249, 7623--7629] in vivo involves transamination with 2-oxoglutarate to produce glutamate, which is then transaminated with pyruvate to produce alanine. The dehydrogenase activity assayed by the decarboxylation of methyl-2-oxo[1-14C]pentanoate was localized exclusively in the fraction containing mitochondria and required NAD+, CoA and thiamin pyrophosphate for optimal activity. Measurements of competitive inhibition suggested that the oxo acids of leucine, isoleucine and valine are all decarboxylated by the same enzyme. The enzyme activity was decreased by 90% upon freezing or sonication and was stimulated severalfold by Mg2+, K+ and phosphate ions. In addition, it was markedly inhibited by ATP, but not by non-metabolizable analogues. This observation suggests that splitting of ATP is required for inhibition. The oxidative decarboxylation of 4-methyl-2-oxopentanoate by the dehydrogenase appears to be the rate-limiting step for leucine oxidation in muscle homogenates and also in intact tissues. In fact, rat muscles incubated with [1-14C]leucine release 1-14C-labelled oxo acid into the medium at rates comparable with the rate of decarboxylation. Intact muscles also released the oxo acids of [1-14C]valine or [1-14C]isoleucine, but not of other amino acids. These findings suggest that muscle is the primary source of the branched-chain oxo acids found in the blood.


Cycloheximide as a probe of fibrinogen synthesis.

Fibrinogen synthesis in the intact rat was perturbed by treatment with cycloheximide. Specific radioactivities of fibrinogen in plasma and liver both decreased at 2 h after treatment and increased over 2-fold by 18 h. Labelled-antibody--polyribosome binding experiments showed that more polyribosomes were engaged in fibrinogen synthesis at 18 h after treatment. Radioactivity of plasma fibrinogen chains from untreated control rats showed a constant ratio of A alpha--B beta/gamma = 1.03. At 2 h after cycloheximide treatment the A alpha- and B beta-chains showed the greatest decrease in labelling (A alpha--B beta/gamma = 0.66) and at 18 h all chains were much more labelled (the A alpha--B beta/gamma ratio chainged to 1.39). The observed imbalance in fibrinogen-chain synthesis suggests that cycloheximide has a selective effect on gene expression.


The influence of thyroxine administered in vivo on the transmembrane protonic electrochemical potential difference in rat liver mitochondria.

When mitochondria from normal and thyroxine-treated rats were energized by incubation with succinate, phosphate and MgCl2, it was found that the hormone treatment increased the transmembrane protonic electrochemical potential difference by 16mV and the respiration rate by 46%. Other experiments show these changes to be associated with increases in the intramitochondrial K+ and phosphate concentrations.


Effects of carbon tetrachloride on isolated rat hepatocytes. Inhibition of protein and lipoprotein secretion.

The effects of carbon tetrachloride on protein and lipoprotein secretion, and on lipid peroxidation, have been investigated in isolated rat hepatocytes. It was found that although the free-radical scavenger promethazine completely suppressed the increased peroxidation produced by carbon tetrachloride, it had no effect on the inhibitory action of carbon tetrachloride on lipoprotein secretion. In consequence, the latter effect of carbon tetrachloride does not appear to be mediated through a peroxidative stage.


Immune complexes in rheumatoid arthritis sera and synovial fluids: a comparison of three methods.

Sera and synovial fluids from 88 patients with rheumatoid arthritis were examined for circulating immune complexes by three assays: monoclonal rheumatoid factor radioimmunoassay, C1q binding assay, and Raji cell radioassay. Paired samples were available for 82 patients. Immune complexes were detected with high frequency in the synovial fluid by each assay (75% by the monoclonal rheumatoid factor radioimmunoassay, 95% by the C1q binding assay, and 61% by the Raji cell readioassay). In rheumatoid arthritis sera, immune complexes were detected with high frequency by the C1q binding assay (85%) and the monoclonal rheumatoid factor radioimmunoassay (70%) but infrequently by the Raji cell radioassay (26%). The presence of immune complexes in serum was most frequently accompanied by the presence of complexes in fluid, regardless of the method of detection; moreover, the levels of immune complexes in synovial fluid were generally higher than in paired serum. Further, the levels of immune complexes as measured by the C1q binding assay correlated with certain parameters of clinical activity, while the monoclonal rheumatoid factor radioimmunoassay and Raji cell radioassay correlated with extraarticular features (excluding nodules) of rheumatoid arthritis.


Increased antibody-dependent cell-mediated cytotoxicity by monocytes from patients with rheumatoid arthritis.

Antibody-dependent cell-mediated cytotoxicity (ADCC) was studied in adult patients with rheumatoid arthritis (RA) and in healthy controls. Three effector cell populations from the peripheral blood were studied which included a mixed mononuclear population, a monocyte-depleted fraction, and a monocyte-enriched fraction. The monocyte-enriched fraction from patients with RA mediated a significantly increased degree of cytotoxicity. Enhanced cytotoxicity was more evident at low effector : target ratios. There was no significant difference in ADCC activity between patient cells and control cells when either the mixed mononuclear population or monocyte-depleted population was studied as effectors. The enhanced effector function of the peripheral blood monocyte in this system may be further indication that mononuclear phagocytes are "activated" in patients with RA.


Development and reversal of a proteoglycan aggregation defect in normal canine knee cartilage after immobilization.

Healthy adult dogs were studied for a defect in proteoglycan aggregation by immobilizing one limb for varying periods of time. Immobilization for 6 days resulted in a 41% reduction in proteoglycan synthesis by articular cartilage from the restrained knee compared with the contralateral control knee. After 3 weeks of immobilization, proteoglycan aggregation was no longer demonstrable in cartilage from the constrained limb. The aggregation defect was rapidly reversible and aggregates were again normal size 2 weeks after removal of a cast that had been worn for 6 weeks.


Congenital contractural arachnodactyly: description of a new kindred.

A previously unreported case of congenital contractural arachnodactyly (CCA) is described. This hereditary connective tissue abnormality resembles Marfan's syndrome in certain respects, but is characterized by camptodactyly rather than joint laxity, as well as by congenital contractural deformities of the knees and elbows. In addition, there is a peculiar, fairly characteristic deformity of the external ear. Like Marfan's syndrome, it is transmitted in autosomal dominant fashion. Despite the superficial skeletal resemblance, however, the cardiovascular and ocular complications of Marfan's do not seem to occur and therefore differentiation of the two syndromes is important.


Synovial fat necrosis associated with ischemic pancreatic disease.

A 59-year-old man with ischemic pancreatic disease, polyarthritis, and cutaneous nodules has shown histopathologic findings indicative of disseminated fat necrosis in a percutaneous biopsy specimen from the right knee. The histopathologic findings in the synovium included necrotic fat cells, distorted fat cells and adjacent lymphocytes, lipid laden histiocytes, and giant cells. In prior histopathologic studies of the joint involvement associated with this disorder, fat cell necrosis has been found only in the periarticular tissues, and the synovium has appeared normal or showed nonspecific inflammation. However, the present study shows that the synovial membrane may also be the site of fat necrosis and an associated inflammatory reaction; thus patients with this disorder may manifest arthritis in addition to periarthritis.


Fluorescein angiography in the surgical treatment of cerebral ischemic lesions.

The present paper summarizes our experiences with intraoperative fluorescein angiography in 50 external carotid artery-internal carotid artery bypass operations. This technique has provided precise localization of the ischemic lesion and aided in selecting the sites and direction of the anastomoses to be performed, as well as in checking the patency of the anastomoses and the area supplied by them. A comparison of X-ray angiography with fluorescein angiography indicates the reliability of the latter in the estimation of the area supplied by the anastomosis.


Eosinophilic fasciitis: a clinicopathologic study.

Eosinophilic fasciitis is a syndrome that is characterized by induration of the skin, peripheral eosinophilia, and a good clinical response to corticosteroid therapy. A skin biopsy specimen from a 69-year-old man who died four months after diagnosis of eosinophilic fasciitis showed that vessel walls in the fascia were invaded by inflammatory cells; endothelial cell proliferation appeared to lead to obliteration of the lumen. No evidence of internal organ involvement was noted at postmortem examination. These findings suggest that, although cutaneous vasculitis may play a role in this disorder, lack of visceral involvement and generally benign course of the disease dictate conservative therapy.


[Activity of postheparin lipoprotein lipase in rabbit blood plasma].

Activity of lipoprotein lipase was studied in rabbit blood plasma after administration of heparin at a dose 50 un/kg into animals. NaCl was added to incubation mixture at final concentration 1 M to differentiate the enzyme from liver triglyceride lipase. The salt inhibited strongly (sometimes completely) the lipolytic activity in rabbit postheparin blood plasma. These data suggest predominance of lipoprotein lipase, sensitive to NaCl, in total lipolytic activity of rabbit postheparin blood plasma. Only slight alterations in the value of total lipolytic activity and in activity, depending on lipoprotein lipase, were observed in rabbits maintained at high cholesterol diet. Under these conditions distinct increase in activity of triglyceride lipase, stable to NaCl , was not found.


Hepatomegaly due to self-induced hyperinsulinism.

Repeated hypoglycaemic attacks, associated with transient hepatomegaly, in a 12-year-old insulin-dependent diabetic girl continued despite reduction in dose and, later, complete discontinuance of insulin. The attacks ceased while she was in hospital, necessitating reinstitution of insulin. The hepatomegaly resolved when surreptitious additional insulin injections were discovered and stopped. Hepatomegaly in diabetics should arouse suspicion of overdosage with insulin.


[The ultrastructural study of choroid plexus papillomas (author's transl)].

Four cases of choroid plexus papilloma (CPP) obtained at the time of surgical excision were examined by light and electron microscopy and compared with normal choroid plexus (CP) of mouse and chick. Ultrastructurally CPPs were the same as CP except in the following two points: In CCPs 1) pinocytotic vesicles of the vascular endothelium and 2) cytoplasmic filaments were more increased in number than in CP. In one case, which showed malignant changes in some parts, we found prominently increased spot desmosomes and cytoplasmic filaments as compared with other three cases, microcysts at the apical portion with other features suggesting the secretory process except for the absence of basal infoldings and myelin bodies. As for the apical tight junction fusion of the two outer-leaflets of the adjacent cytoplasmic membrane was observed as in CP. This fact suggests that there is a blood-CSF barrier also in CPPs as in CP.


[A case of "ping-pong ball eye movement (author's transl)].

We experienced a rare and peculiar eye movement in a comatose patient with ruptured intracranial aneurysm. The patient's eyes moved quickly and irregulary from one extreme position to the other with whirling and bounding components. Such abnormal eye movement occasionally halted, but abruptly reappeared. We named this abnormal eye movement which had never been reported before, "ping-pong eye movement". Neuropathological study revealed a subarachnoid hemorrhage and the diffuse infarction in the right remporal lobe, and a smal clot in the fourth ventricle without specific findings in the brainstem and the cerebellum. Consideration was made that the hemorrhage in the fourth ventricle resulted in a hyper-irritable state in the fastigial nuclei, from which the abnormal discharges were driven to the reticular formation, the vestibular nuclei, the MLF, and the oculomotor complexes. We conclude that this "ping-pong" ball eye movement" might be the appearance of epilepsy in the eyes, due to the epileptogenic focus of the fastigial nuclei.


Measurement of the relative contributions of rib cage and abdomen/diaphragm to tidal breathing in man.

A simple mathematical model of the chest wall was constructed so that during tidal breathing the relative volume contributions of the rib cage and abdomen/diaphragm could be measured in man, using four mercury-in-rubber strain gauges around the trunk. From the dimensions of the trunk and the change in circumference determined by the four gauges, the separate contributions of rib cage and abdomen/diaphragm could be determined using a purpose-built analog computer. The system was evaluated in 13 laboratory personnel, and in 13 other subjects before and after anaesthesia. There was a linear relationship between tidal volumes computed and measured at the mouth, over the residual volume to (FRC + 1 litre) range, with an error of +/- 8%. The relative contribution of rib cage to tidal breathing showed a large scatter from 5 to 42% with a non-significant tendency to decrease with age.


Regional blood flow in normovolaemic and hypovolaemic haemodilution. An experimental study.

The effects on the circulation of limited normovolaemic haemodilution with dextran 70 and subsequent haemorrhage to a mean arterial pressure of 60 mm Hg were studied with isotope-labelled microspheres in the dog. Following haemodilution, cardiac output, stroke volume and systemic oxygen transport increased. The distribution of oxygen to the heart, liver (hepatic artery), spleen and carcass (mainly muscle, skeleton and skin) was increased, while a decrease in oxygen supply to the brain was found. Following haemodilution and haemorrhage, cardiac output, systemic oxygen transport and mixed venous oxygen tension decreased. Blood flow was redistributed to maintain the cerebral, renal, hepatic arterial and coronary circulations, mainly at the expense of blood flow to the carcass and through systemic arterio-venous shunts. Thus, limited normovolaemic haemodilution does not affect the normal circulatory response to moderate haemorrhagic hypotension.


Acute and subchronic neuromuscular blocking characteristics of streptomycin: a comparison with neomycin.

The characteristics of the neuromuscular block produced by streptomycin in vivo were studied on the sciatic-tibialis anterior nerve-muscle preparation of eight anaesthetized cats. The lungs of the animals were ventilated mechanically and normocarbia was maintained. During acute exposure to streptomycin (within 2 h), ED50 for blockade of the twitch was 56 (SEM +/- 5) mg kg-1 of the base. The characteristics of block were similar to those of neomycin-induced block in some aspects. There was absence of train-of-four fade and tetanic fade, partial sparing of the responses elicited at 10 Hz and 20 Hz, and total sparing of the 50 Hz tetanus, as well as the post-tetanic twitch. In contrast to neomycin-induced neuromuscular block, however, post-tetanic exhaustion was not observed and prolonged exposure to streptomycin (22-28 h) did not change the characteristics of the block. We conclude that, despite their chemical similarities, streptomycin and neomycin block neuromuscular transmission differently.


Effects of ageing on the pharmacokinetics of pancuronium.

The effects of age on the pharmacokinetics of pancuronium were investigated. The distribution volume of pancuronium did not appear to be age-dependent, but elimination of the drug decreased with increasing age. The clinical implications of these findings are discussed.


Prevention of suxamethonium-induced changes in serum potassium concentration by hexafluorenium. Is their combined use justified?

Sixty patients, none of whom was suffering from renal failure, received neurolept anaesthesia. They were divided into six groups of 10 patients each. Groups I and IV, II and V, and III and VI were given suxamethonium 0.2, 0.6 and 1.0 mg kg-1 respectively. Groups IV-VI were pretreated with hexafluorenium 0.3 mg kg-1. The serum potassium concentration decreased significantly after the induction of anaesthesia and also following the administration of hexafluorenium. Neither suxamethonium 0.2 mg nor 0.6 mg kg-1 with or without hexafluorenium restored the potassium concentration to the control value. Suxamethonium 1.0 mg kg-1 alone caused the serum potassium to increase to values greater than control; hexafluorenium attenuated this effect. The combination of hexafluorenium and suxamethonium may be of benefit in patients who are anephric or are in chronic renal failure.


Morbidity of day-case gynaecological surgery. A comparison of thiopentone and Althesin.

To compare two anaesthetic techniques, morbidity following day-case gynaecological surgery in 170 patients was assessed by means of a questionnaire at the time of leaving hospital. A further questionnaire sought information for the evening of the operation, and the 1st and 2nd days after operation. Ninety-four per cent of patients returned the latter questionnaire. There was a high frequency of morbidity extending into the 2nd day after operation. An anaesthetic technique using Althesin and nitrous oxide in oxygen offered no advantage over a technique with thiopentone and nitrous oxide in oxygen and halothane.


Haemoptysis following insertion of a Swan-Ganz catheter.

Haemoptysis occurred in a patient in whom a Swan-Ganz catheter was inserted for the induction of anaesthesia for hemicolectomy. It is suggested that acute pulmonary hypertension, superimposed on existing chronic pulmonary hypertension, superimposed on existing chronic pulmonary hypertension associated with mitral stenosis, was an important aetiological factor in the haemoptysis.


Congenital fibro-osseous dysplasia of jaws ("hippopotamus face"). An anaesthetic problem.

Two brothers with "hippopotamus face" deformities presented for corrective surgery under general anaesthesia. In the first patient, blind nasal intubation was successful. In the second patient, tracheotomy was avoided by the use of an intubating bronchoscope.


Kinetics of fluphenazine after fluphenazine dihydrochloride, enanthate and decanoate administration to man.

1 Fluphenazine (1,2-ethanol 14C) was administered to seven human subjects as the dihydrochloride and as the enanthate and decanoate esters. 2 The subjects had previously been treated with fluphenazine injections for at least 6 months. 3 Fluphenazine was separated from its radioactive metabolites by selective solvent extraction. Plasma concentrations were measured for up to 21 days after dosing. 4 The preparations showed differences in peak concentrations, times of the peaks and half-times of the elimination phase. The longest half-times occurred with the decanoate and the shortest with the dihydrochloride. 5 It is postulated that the differences in kinetics relate principally to the release of the compound from the site of injection. 6 There was no evidence for presence of the esters in plasma, urine or faeces.


Specific radioimmunoassay of amitriptyline and nortriptyline.

1 Antisera to nortriptyline were prepared by immunizing rabbits with N-succinylnortriptyline--bovine serum albumin conjugate. 2 A sensitive radioimmunoassay has been developed for the tricyclic antidepressants amitriptyline and nortriptyline. 3 amitriptyline and nortriptyline are separated from each other and from interfering metabolites before assay. 4 Using [3H]-imipramine and [3H]-succinylnortriptyline as tracers the radioimmunoassay can measure amitriptyline and nortriptyline levels down to 2--3 ng/ml using 0.05 ml plasma sample. 5 Agreement between the radioimmunoassay and a gas-chromatographic assay was excellent for both amitriptyline and nortriptyline.


Plasma cinnarizine levels resulting from oral administration as capsule or tablet formulation investigated by gas-liquid chromatography.

1 A gas chromatographic assay for the estimation of therapeutic concentrations of cinnarizine in plasma is described. 2 Cinnarizine (75 mg) was administered orally to twelve healthy subjects in the form of capsules and tablets on two separate occasions. No difference was found in the plasma levels or absorption of cinnarizine from these formulations. 3 The mean plasma elimination half-life of cinnarizine was 3.24 h.


A comparison of some extra-renal effects of spironolactone and canrenone.

1 Measurement of changes in trans-mural rectal potential difference (t.m.r.p.d.) and plasma aldosterone levels have been used in a comparison of the extra-renal activities of spironolactone and its major metabolite canrenone. 2 A characteristic pressure artifact was observed during measurement of t.m.r.p.d. When pressure artifacts were eliminated, there was a log-linear relationship between increasing doses of intravenous aldosterone and maximum increase in t.m.r.p.d. 3 Pre-treatment for 5 days with spironolactone or canrenone produced a similar attenuation of the increase in t.m.r.p.d. produced by infused aldosterone, suggesting that canrenone is the active metabolite of spironolactone in the rectum. This is in contrast to the significantly greater renal activity of spironolactone that has been demonstrated after a similar treatment period. Neither antagonist treatment produced significant changes in pre-infusion plasma aldosterone concentrations. 4 The need for assay of the extra-renal activities of aldosterone antagonists in the assessment of their therapeutic potential is discussed.


Pituitary tumours during pregnancy in mothers treated with bromocriptine.

1 Out of 805 previously infertile women in whom pregnancy was achieved on bromocriptine treatment, 137 were diagnosed as having pituitary tumours. 2 In nine of these, tumour-related complications occurred during pregnancy, chiefly visual field impairment. Surgical intervention was considered necessary in two patients. In a third patient reinstitution of bromocriptine produced remission of symptoms. 3 Although the frequency of serious complications was low, the present state of knowledge indicates that surgery or irradiation of pituitary tumours should be preferred, at least as the first line of treatment, for patients contemplating pregnancy.


Concurrent administration of antacids and prednisone: effect on serum levels of prednisolone.

1 Specific radioimmunoassays have been developed for the measurement of prednisolone and prednisone in serum. 2 Serum prednisolone levels have been measured following the administration of prednisone both with and without antacids. 3 There were no significant differences in the values of Cmax (mean +/- s.d.) (251 +/- 80 ng/ml with, 245 +/- 69 ng/ml without antacid), AUC (1430 +/- 469 ng ml-1 h-1 with, 1406 +/- 530 ng ml-1 h-1 without antacid) or Tmax (1.6 +/- 0.6 h with, 1.6 +/- 0.8 h without antacid). 4 Serum prednisone levels were less than 20% of prednisolone levels at all time intervals in the subjects studied. 5 Concurrent antacid administration has no significant effect on serum prednisolone levels attained.


Biological activities of dihydrodiols derived from two polycyclic hydrocarbons in rodent test systems.

Comparisons have been made between (a) the initiation of tumours in mouse skin, (b) the induction of hyperplasia and the suppression of sebaceous glands in mouse skin and (c) the induction of s.c. tumours in rats, by either benzo[a]pyrene or 7-methylbenz[a]anthracene and their related K-region and non-K-region dihydrodiols. Whilst the 3,4-dihydrodiol derived from 7-methylbenz[a]anthracene is more active than the hydrocarbon in initiating tumours in mouse skin (subsequently promoted by a phorbol ester) the 7,8-dihydrodiol of benzo[a]pyrene is very much less active than benzo[a]pyrene itself in the induction of hyperplasia or the suppression of sebaceous glands in mouse skin or in the induction of s.c. sarcomas in rats. Since much other evidence suggests that the 3,4-dihydrodiol of 7-methylbenz[a]anthracene and the 7,8-dihydrodiol of benzo[a]pyrene are the dihydrodiols involved, via the related vicinal diol-epoxides, in the metabolic activation of these hydrocarbons, mouse skin initiation-promotion experiments may be more useful for the identification of such diols than the other two in vivo tests for biological activity used here.


DNA fragmentation in some organs of rats and mice treated with cycasin.

Cycasin (methylazoxymethanol-beta-D-glucoside) is carcinogenic in several animal species. It produces a variety of malignant tumours, mainly in the liver of mice, and in the liver, kidney and large intestine in rats. It does not appear to be mutagenic in the Ames test, even in the presence of liver microsome fraction, and it is among those carcinogens (less than 10%) ranked as "false negatives" in this test. The ability of cycasin to damage in vivo liver, kidney, lung and colonic DNA of Wistar rats and C57BL/L mice was investigated by means of alkaline elution technique. Oral single-dose administration of cycasin, in the range of 50-400 mg/kg body weight, produced in the rat a clearly evident dose-dependent DNA fragmentation in the liver, and less marked damage to DNA from kidney and colon mucosa. In mice, the same treatment produced dose-dependent DNA damage only in the liver. DNA repair up to 18 h appeared to be incomplete both in mice and rats. Methylazoxymethanol acetate is considered to be an active form of cycasin. While in vivo methylazoxymethanol acetate caused DNA damage, in vitro it appeared inactive and required metabolic activation, possibly consisting in its hydrolysis by esterase activity, to be able to cause DNA fragmentation.


Cell-cycle inhibitory effects of the mitotic inhibitor NY 3170 on human cells in vitro.

Effects of the mitotic inhibitor NY 3170 (1-propargyl-5-chloropyrimidin-2-one) on cell-cycle kinetics of NHIK 3025 cells were studied by means of time-lapse microcinematography, pulsed incorporation of [3H] thymidine, flow cytometry, and mitotic index. All the experiments were performed with cells synchronized by mitotic selection. Mitotic inhibition as well as inhibition in interphase was examined. The small fraction of cells able to escape mitotic arrest at 0.2mM NY 3170 had spent about 12 h in metaphase. The metaphase block was complete at 0.3 mM. For comparison, complete metaphase arrest of NHIK 3025 cells was reached at 8 mM after treatment with the parent substance NY 3000 (5-chloropyrimidin-2-one, previously reported). At 0.3mM NY 3170 interphase was also considerably prolonged. All stages of interphase were prolonged, in contrast to the interphase prolongation after treatment with high concentrations of the mitotic inhibitors vincristine and vinblastine, which occurs in G2. It was shown that the presence of NY 3170 during mitosis is a necessary and sufficient condition for metaphase arrest, thus demonstrating that metaphase arrest is not dependent on some preceding event in interphase.


Distribution and tumour cytotoxicity of the radiosensitizer misonidazole (Ro-07-0582) in C57 mice.

The distribution and clearance of misonidazole (MIS = Ro-07-0582) were studied in C57 mouse tissues and in transplants of Lewis lung tumour. The half life of the drug in blood after a dose of 1 mg/g i.p. was 3 h. Some tissues, such as liver, were found to have consistently low MIS levels, and this was found to be due to degradation of the drug after removal of the tissues from the host. The in vivo cytotoxicity of MIS to Lewis lung tumour cells was studied using an in-vitro colony assay. After half of the tumours had been irradiated with 10 Gy to kill most of the oxic cells, the mice received i.p. injections of MIS. To simulate the longer drug exposure of human tumour cells (due to the longer half life in man) a repeated injection regime was used in some mice. There was no significant cell kill after a single dose, but with a prolonged exposure to the drug in the multiply injected animals, cell survival was reduced to 50% of control in both the irradiated and unirradiated tumours. Since the hypoxic fraction of the unirradiated tumour is probably not more than 30%, it would appear that MIS is not selectively cytotoxic to hypoxic cells. However, MIS had a much greater cytotoxic effect upon hypoxic Lewis lung tumour cells in vitro, with very little or no effect on cells grown in air. This would support the theory that the presence of hypoxic cells is essential for the expression of MIS cytotoxicity.


Lack of effect of immunotherapy with BCG and Corynebacterium parvum on hepatic drug hydroxylation in man.

Serial serum diphenylhydantoin and urinary 5-(p-hydroxphenyl)-5-phenylhydantoin concentrations were determined in 8 patients with malignant disease and 4 healthy volunteers on 2 separate occasions after an oral dose of diphenylhydantoin (500 mg). No significant difference was observed between metabolism before and 10 days after immunization with BCG or Corynebacterium parvum. Volunteers without intervening immunization similarly showed no difference.


Microenvironmental arginine depletion by macrophages in vivo.

Since the tumour-selective cytotoxic activity of activated macrophages in vitro can be attributed to depletion of the culture medium of L-arginine by macrophage arginase, a series of experiments was designed to determine whether such a mechanism could operate in vivo. Extracellular fluid obtained from Gullino chambers within established tumours contained high levels of arginase, no detectable arginine and high levels of ornithine. When tumours were disaggregated into single-cell suspensions, arginase was readily detected within tumour macrophages but not within malignant cells. Inflammatory ascites induced in mice by Corynebacterium parvum was rich in arginase, depleted of L-arginine and cytotoxic in vitro to L5178Y and V79 cells. High levels of arginase in the ascites fluid were associated with resistance to challenge with syngeneic L5178Y cells. Lymph collected from the cisterna chyli in rats bearing a macrophage-rich sarcoma on the small bowel contained elevated levels of arginase, was depleted of arginine and contained increased concentrations of ornithine. We conclude that in sites of macrophage infiltration there is microenvironmental arginine depletion due to the action of arginase, and that arginase release could represent an important macrophage effector mechanism against a variety of targets, including malignant cells, virus-infected cells, fungi and parasites.


Effects of carrageenan, PVP and tumour-bearer serum on immunity induced by excision or mitomycin C-treated tumour cells in mice.

Carrageenan (Cg) was tested for its effects on the growth of, and immunity to, 2 methylcholanthrene-induced syngeneic murine fibrosarcomas (H1 and H2). The tumours were found not to share major tumour-specific transplantation antigens. H2 appeared more immunogenic than H1. In contrast to H1, immunity induced by H2 was not affected by Cg, nor was its growth in Cg-treated normal mice augmented.Postoperative i.p. injections of Cg abolished the weak anti-H1 immunity produced by H1 tumour excision. Furthermore, the subsequent growth of the H1 tumour challenge in the Cg-treated immune mice was significantly greater than the augmented growth in Cg-treated normal mice. The Prior administration of the macrophage-stabilizing agent polyvinylpyrrolidone (PVP) to immune mice significantly reduced the augmenting effect of Cg. The growth-promoting effect of Cg on a secondary H1 tumour challenge in mice immunized by tumour excision was abolished by 10(6) MCT-H1 cells injected s.c. before Cg. In contrast to the immunity induced by tumour excision, Cg did not abolish the immunity induced by the injection of MCT-H1 cells.Passive administration of H1 tumour-bearer serum (TBS) did not enhance the growth of H1 cells in normal mice, nor did TBS abrogate the specific cell-mediated immunity (CMI) induced in vivo by MCT-H1 cells. However, TBS administered to Cg-treated, MCT-H1-immune mice abolished tumour immunity.We propose that TBS does not inhibit CMI in vivo provided that macrophages remain functional, but may do so when macrophages are rendered defective by antimacrophage agents or by products of neoplastic cells. Increasing the levels of specific effector cells can over-ride the inhibiting effects of TBS, even when defective macrophages are present.


Antibody-dependent and spontaneous lympholysis in urologic cancer patients.

To evaluate cytotoxic function mediated by killer lymphocytes and macrophages in urological cancer patients, we examined antibody-dependent and spontaneous lympholysis of chicken erythrocyte target cells, which is mediated by macrophages. Our results demonstrate a discordance between cytotoxic mechanisms in cancer patients, killer-cell function being impaired whilst macrophage-mediated cytotoxicity was increased.


Effect of treatment on the immunological status of women with advanced breast cancer.

An immunological profile has been serially studied in 72 patients with advanced breast cancer during the course of a randomized trial of chemotherapy and hormonal manipulation. DNCB+ patients were more likely to respond to either therapy, but no other test was predictive of response. In the follow-up period all chemotherapy patients had a reduction in white-cell count which was significantly greater in those responding to treatment. None of the other tests (phytohaemagglutinin response, immunoglobulins G, A and M, or Mantoux test) demonstrated changes that could be related to treatment or response, but there was a gradual unexplained fall in IgM levels in all groups the study progressed. It is concluded that the chemotherapeutic regimen (cyclophosphamide, vincristine, adriamycin and 5-fluorouracil) is relatively non-immunosuppressive, and that hormonal therapy (oophorectomy, tamoxifen or androgens) had no detectable effect on the immune response.


Lipogenetic and glycolytic enzyme activities in carcinoma and nonmalignant diseases of the human breast.

Activities of some enzymes associated with carbohydrate and lipid metabolism were determined in 48 human breast carcinomas and compared with those found in 35 nonmalignant breast tumours and also in 13 normal breast tissues. In fibrocystic disease only the activity of citrate lyase was markedly higher (14-fold) than in normal tissue. The activities of the remaining enzymes did not differ significantly from those in normal tissue. Enzyme activities in breast carcinoma were 4--160 x those determined in normal tissue according to the following sequence : phosphofructokinase less than malate NADP dehydrogenase less than hexokinase less than lactate dehydrogenase less than isocitrate NADP dehydrogenase less than ATP citrate lyase. Activity of citrate lyase, very low in normal breast (0.0017 mumol/min/g of tissue) rose gradually to 0.039, 0.072 and 0.258 mumol/min/g of tissue in localized fibrocystic disease, fibroadenomas and carcinomas respectively. These data support the idea that citrate lyase may play an important role in lipogenesis in hyperplastic human breast tissues.


Ratio of 11-desoxy 17-oxosteroids to creatinine in a population screened for breast cancer.

During a population-based screening project for breast cancer, almost 15,000 women aged 50 years and over have provided a 12 h (overnight) sample of urine for research purposes. In 3,789 women the excretion of 11-desoxy-17-oxosteroids (DOOS) and creatinine was measured. Results were analysed in terms of urinary concentrations and of a ratio between DOOS and creatinine. Age had an effect on DOOS, creatinine and their ratio. Body weight and body surface area had an effect on creatinine excretion and therefore on the ratio. The following variables did not have an appreciable effect on the above-mentioned ratio: a family history of breast cancer, parity and age at first pregnancy, menopause and oestrogenic drugs, and parenchymal pattern of the breast as observed on the xeromammogram. Breast cancer was found at first screening in 106 out of 14,697 women. In 100 of these cases DOOS and creatinine were measured. Excretion values expressed as the ratio between the two, allowing for body surface area, did not differ materially from those of 100 age-matched controls. These results lead the authors to the conclusion that the determination of androgen metabolite excretion in women over 50 years of age is of no help in selecting a group at high risk of breast cancer.


Serum testosterone and dihydrotestosterone in carcinoma of the prostate.

Serum testosterone (T) and dihydrotestosterone (DHT) were measured by a sensitive and reliable radioimmunoassay in 42 normal subjects and 33 age-matched patients with carcinoma of the prostate. The mean +/- s.e. for serum testosterone in normal subjects was 16.74 +/- 0.76nM and the corresponding value for patients with carcinoma was 20.94 +/- 1.48nM. Statistical analysis of the results showed a significant increase in T level in patients with carcinoma of the prostate (P less than 0.01). In contrast, there was no difference in DHT concentration between the two groups, values being 2.43 +/- 0.09 and 2.06 +/- 0.09nM for normal subjects and patients respectively. The means +/- s.e. for T/DHT ratio in controls and patients were 6.8 +/- 0.2 and 12.8 +/- 1.3 respectively. The difference was highly significant (P less than 0.001). The wide range of variation for T in patients with carcinoma would suggest that although mean T is higher in these patients, this measurement alone is of little practical value, whereas T/DHT ratio is a more reliable parameter in evaluating the androgen changes in these patients. The significance of these findings in relation to the aetiology of the disease is discussed.


Case-control study: soft-tissue sarcomas and exposure to phenoxyacetic acids or chlorophenols.

In 1977 a number of patients with soft-tissue sarcomas and previous exposure to phenoxyacetic acids were described. Following from these observations a matched case-control study was made. Exposure to chlorophenols was also included in this study. The results showed that exposure to phenoxyacetic acids or chlorophenols gave an approximately 6-fold increase in the risk for this type of tumour. It was not possible to determine, however, whether the carcinogenic effect was exerted by these compounds or by impurities such as chlorinated dibenzodioxins and dibenzofurans that in almost all cases were part of the commercial preparations.


Diet, liver function and dimethylhydrazine-induced gastrointestinal tumours in male Wistar rats.

Male Wistar rats fed a normal laboratory pelleted diet, when treated s.c. with 1,2-dimethylhydrazine (DMH) 10 mg/kg/wk survived the 24-week experiment, showed no signs of chemical toxicity or macroscopic liver damage, and developed mainly large-bowel tumours. Conversely, male Wistar rats treated with 20 mg/kg/wk DMH did not survive the full term of the experiment and developed ascites, pleural effusions and nodular livers. They also developed more small-bowel tumours than large-bowel tumours. The relationship between the predominant site of tumour development and dosage of DMH was highly significant.Male Wistar rats fed with an all-liquid diet (Vivonex) and treated with 20 mg/kg/wk DMH behaved quite differently both in terms of survival and site of tumour development. These rats survived the full term of the experiment, showed no signs of chemical toxicity, experienced minimal liver damage and developed predominantly large-bowel tumours. The protection afforded by the all-liquid diet against DMH toxicity and small-bowel tumour induction was statistically highly significant.A series of blood tests with special reference to liver function confirmed the highly significant degree of protection against liver damage afforded by the all-liquid diet.Sections of liver from treated rats were examined, and a simple pathological scoring system was devised which showed a highly significant difference in liver histology between standard diet and liquid-diet rats treated with 20 mg/kg/wk DMH.The results strongly suggest an association between severity of liver damage from DMH and the subsequent development of small-bowel tumours. The all-liquid diet protected rats from liver damage and these rats developed significantly fewer small-bowel tumours.


Metastatic colonization potential of primary tumour cells in mice.

A model has been developed for studying the capability of cells from primary murine mammary tumours to establish colonies in distant organs. The model involves the i.v. inoculation of disaggregated tumour cells into autologous and syngeneic recipients. The results show that the metastatic colonization potential of cells from a given tumour is consistent within the animals of an inoculated batch. Also, the findings are uniform in the autologous host and the syngeneic recipients. Tumours vary in their colonization potential and can be classified in 2 main groups designated high and low. These findings indicate that: (i) cells from 37% of mammary tumours can heavily colonize the lungs when inoculated i.v., even though the incidence of metastatic spread of these tumours in the undisturbed animal is almost zero. Thus, the relative infrequency of spontaneous metastasis from murine mammary tumours is not due to inability of the tumour cells to survive and colonize once free in the blood stream; and (ii) the colonization potential of the tumours is an intrinsic property of the tumour cells rather than of the host, whose prior acquaintance with the cells does not seem to confer resistance to colonization. The model presents opportunities for identification of possible differences between tumours of high and low colonization potential, and is being used to study cellular properties which favour colonization of distant organs by comparison of observations in vitro with the behaviour of cells from the same tumour in vivo.


Cellular progression of neoplasia in the subcutis of mice after implantation of 3,4-benzpyrene.

An implantation model has been used to investigate the cellular progression of chemically induced subcutaneous neoplasia in the mouse. Implantation of 3,4-benzpyrene induced persistent changes in the normal process of connective tissue formation around the implant. Light-microscope and autoradiographic studies have shown a temporal progression from aberrant filter- or muscle-associated cells through proliferative foci to large invasive sarcoma. Electron microscopy revealed that presarcomatous cell foci consisted of one of two different cell types. These were either spindle cells with ultrastructural characteristics similar to foreign-body-induced sarcoma, or cells with the ultrastructural features of rhabdomyosarcoma. The subsequent appearance of two histological groups of sarcoma that were ultrastructurally similar to the cells of the early proliferative foci indicated that both elements may progress to form tumours. However, the constituent cells of both groups of tumours displayed a broad histological and ultrastructural spectrum and the marked similarity between the undifferentiated cells of each suggested that both may have arisen from diverse differentiation of a common pluripotential cell such as the pericyte.


Neural changes in recurrent genital infection of infectious bovine rhinotracheitis virus in calves treated with dexamethasone.

Recurrent genital infection with infectious bovine rhinotracheitis (IBR) virus was induced in calves by dexamethasone (DM) treatment 3 months after primary infection. The virus was first recovered from vaginal secretions on the 3rd day after the initiation of DM treatment; however, the virus was not recovered at all from nasal secretions or from the cerebrospinal fluid of the DM-treated calves. The significant neural changes in all recurrent infected calves were nonsuppurative poliomyelitis in the lumbosacral spinal cords and their ganglia, and they were more severe in the sacrospinal cords than in other parts of the spinal cord. The virus antigen was detected in the sacrospinal ganglia and peripheral nerve fibres in the submucosa of the vagina by the fluorescent antibody technique on the 4th and 6th days after the start of DM treatment. These observations indicated that nonsuppurative poliomyelitis may be a characteristic lesion in recurrent genital infection of IBR virus. The sacrospinal cords and ganglia are considered as latent sites of IBR virus.


Ultrastructural changes in the renal papillary cells of rats during maintenance and repair of profound potassium depletion.

In rats with long-term diet-induced potassium depletion, the cytoplasm of markedly enlarged papillary cells was mainly occupied by membrane-bound droplets, many of which acquired massive proportions. Both free and attached ribosomes were decreased, the Golgi apparatus was inconspicuous and there was a paucity of mitochondria. Despite the overwhelming accumulation of droplets with concomitant loss of normal metabolic organelles, cell death did not occur. With potassium repletion, the organelles readily proliferated regardless of the progression of droplet dissolution. The shrinkage of the droplets was accompanied either by disintegration of the limiting membrane or by disappearance of the constituents within an intact membrane. Microtubules were conspicuous in many of the cells undergoing gradual reduction in size. These cytoplasmic changes in renal medullary cells of rats during long-term potassium depletion and immediate post-repletion periods essentially represented the consequences of maintenance and repair of a storage process.


Ultrastructure of rat lungs following exposure to aerosols of dibenzoxazepine (CR).

Three groups of 18 animals were exposed respectively to the following large doses of dibenz (b.f)-1:4 oxazepine (CR) aerosols, 78,200,140,900 and 161,300 mg/min/m3. Animals were killed at intervals from 15 min to 2 days, and the lungs examined macroscopically, by electron microscopy and conventional histology. There were no deaths during or after exposure. Macroscopically the lungs from all rats appeared normal. Microscopically there were a few areas of mild congestion, haemorrhage and emphysema, but there was little variation between the different groups. Electron micrographs revealed some morphological alteration of the epithelium and endothelium but only occasional changes in the interstitium. The alterations took the form of "ballooning" of the endothelium with isolated foci of swelling and thickening of the epithelium. Interstitial oedema was observed in one animal only which was exposed to the highest concentration. The effects appeared similar in all groups, and are thought to be transient. The results of this investigation suggest that even high doses of CR aerosols cause minimal damage to the lung, and the structural alterations which do occur are believed to be due to the stress to which the animals were subjected during the exposure period.


Liposome-incorporated corticosteroids: I. The interaction of liposomal cortisol palmitate with inflammatory synovial membrane.

Cortisol palmitate-containing liposomes were stable in rheumatoid synovial fluid at 37 degrees. Two hours after the intra-articular injection of such liposomes into experimentally arthritic rabbits relatively high proportions of the steroid and lecithin components were recovered from the synovialis. The level of cortisol in the tissue at this time was inversely related to the chronicity of the inflammation. It was found also that the degree and duration of the anti-inflammatory activity of the liposomes was greatest in the initial acute phases of the inflammation.


The NBT test in burned patients.

The number of polymorphs which stained with the dye nitro-blue tetrazolium (NBT "Positive") increased sharply during the first week after burning, reaching levels 4--5 times above values for healthy volunteers. In burns of more than 20% of the body surface a second, smaller increase in the number of NBT "positives" occurred 4 to 6 weeks after burning. The high levels of NBT "positive" polymorphs occurred independently of infection on the burns. A burned patient who died from septicaemia had very low numbers of NBT "positive" polymorphs for 3 weeks before death.


The initiation of epidermal wound healing in cuts and burns.

An apparatus to produce shallow linear burns is described. The burns contained about the same amount of damaged tissue as a shallow cut. The initiation of healing in cuts and burns on guinea-pig ears was studied using an 3H-thymidine and autoradiographic technique to determine the number of cells in DNA synthesis in epidermis adjacent to the wound. Cuts caused epidermal cells to enter DNA synthesis rapidly; the maximum response was 1 day after injury. Thus response was delayed in burns and the maximum was not seen until at least 3 days after burning. Extracts of normal skin stimulated epidermal DNA synthesis, whereas extracts of burned skin did not. Possible mechanisms affecting the delay in the healing of burns are discussed.


Study of allergenic fungi by means of a laser beam.

Laser microscopy was employed to detect some elements of Mendeleev's classification in allergenic moulds for humans. It permits the detection of 74 elements in the periodic system, from lithium (3) to uranium (92). This method requires little specimen preparation and has the advantage of not requiring large masses of tissue.


Protection of pregnant mice with phospholipase C and with Arvin against foetal death induced by bacterial lipopolysaccharide.

Injection of bacterial lipopolysaccharide into pregnant mice resulted in fibrinogen accumulation, thrombosis and haemorrhage in the placental tissue and foetal death. Depletion of circulating fibrinogen by a thrombin-like enzyme from the venom of Malayan pit viper, Arvin, prevents foetal death. Foetal protection was also obtained by treating the mothers with a preparation of phospholipase C from Bacillus cereus known to inactivate tissue thromboplastin. It is suggested the lipopolysaccharide causes foetal death by inducing thrombosis as a consequence of activation of placental thromboplastin.


The study of biogenetic pathways using a perfusion technique containing perfluorochemicals.

A liver perfusion system was assembled and adapted for pulse labelling studies. The perfusion medium was prepared by emulsifying perfluorotributylamine and Pluronic F 68 in a CO2 atmosphere using a sonicator. Ribosome-rich and ribosome-poor rough microsomes, smooth microsomes and Golgi membranes could be prepared from perfused livers with a good purity and recovery as from non-perfused livers. The subfractionation technique used was simple and involved slight modifications of the methods described earlier by Eriksson (1973) and Ehrenreich et al. (1973). The specific activity of NADPH-cytochrome c reductase in microsomes and of UDP-galactosyltransferase in Golgi membranes from perfused and non-perfused livers were identical. The specific activity of glucose-6-phosphatase in microsomes was slightly decreased after perfusion, but the membrane permeability barrier to glucose-6-phosphate remained intact. The granulated microsomal fractions from perfused liver had a somewhat reduced number of membrane-bound ribosomes. The system developed proved useful in studies of the synthesis and intracellular transport of albumin. The technique should also be suitable for use in studies of membrane biogenesis.


Focal mesangial proliferative glomerulonephritis in the rat caused by Habu snake venom: the role of platelets.

Platelet aggregates are a prominent early feature in the glomerular lesions of focal mesangial proliferative glomerulonephritis induced by Habu venom. After venom injection the peripheral blood platelet count falls rapidly, and in vitro the venom causes platelet aggregation and release of 5-HT. The role of platelets in this model has been studied in rats depleted of platelets by antiplatelet serum administration. No effect on the early (24 h) glomerular damage was found. Mesangial proliferation which appears 2--3 days after glomerular injury was significantly inhibited in platelet-depleted rats. These results show that platelet activity does not initiate the mesangial injury in this model, but suggest that platelets or their products act as a stimulus to mesangial proliferation.


Myocardial capillary density and muscle fibre size in rats born and raised at simulated high altitude.

Rats were born in a hypobaric chamber at a pressure of 500 mmHg and kept there at 460 mmHg for 5, 9 or 13 weeks. The myocardial capillary bed was labelled by perfusion with India ink. The density of myocardial capillaries and the area of cross-section of myofibres was estimated from histological sections. In the left ventricle there was a steady reduction in capillary density with increasing age which was similar in test and control rats. There was also an increase in myofibre size which was similar in test and controls. Hypoxia therefore failed to induce capillary proliferation or a reduction in myofibre diameter in the left ventricle and this is reflected in a constant capillary:fibre ratio. In the right ventricle, ventricular hypertrophy in the hypoxic rats induced a striking reduction in capillary density due to a two-fold increase in myofibre size. There was nevertheless some degree of capillary proliferation, probably a consequence of hypertrophy rather than the direct influence of hypoxia.


Plasma complement and histamine changes in atopic dermatitis.

Fifteen patients with atopic dermatitis were investigated to evaluate the total of complement and histamine. In five patients total serum complement haemolytic activity (CH50) was significantly decreased as was the haemolytic activity of complement components C2 (C2H50) and C3 (C3H50). By counter immunoelectrophoresis split products of C3 were detected. There was no evidence for alternative pathway activation or the presence of an activator of the alternative pathway. In three patients plasma histamine concentrations were elevated. The intensity of the complement and histamine changes observed seemed to be correlated to the severity of the disease.


Ia antigens on indeterminate cells of the epidermis: immunoelectronmicroscopic studies of surface antigens.

An antiserum against human B-lymphoblastoid cell membrane alloantigens (Ia-like antigens) was used to study the presence of such antigens on dendritic cells in human epidermis. Only Langerhans cells and the majority (85%) of so-called indeterminate cells were positively stained, as shown by immuno-electron microscopy. Fifteen percent of the indeterminate cells were negative and were considered to be immature melanocytes. A relationship exists between the indeterminate cell and the Langerhans cell. A proposal is made concerning emigration of Langerhans cells in response to haptenic stimulation, and the immigration of indeterminate cells to restore the status quo.


Disseminated intravascular coagulation associated with toxic epidermal necrolysis (Lyell's syndrome).

Evidence of disseminated intravascular coagulation was recorded in eight patients with toxic epidermal necrolysis (TEN)--Lyell's syndrome. Patients were treated with low doses of heparin in combination with the usual treatment of TEN, i.e. maintenance of fluid and electrolyte balance, systemic corticosteroids, antibiotics and aseptic dressings, in the Intensive Care Unit environment. It is suggested that the alteration of haemostasis and inter-related biological systems, such as activation of components of complement, kinins and immunoglobulins, may affect the outcome of TEN.


Erosive pustular dermatosis of the scalp.

We report six patients with a previously undescribed but characteristic pustular dermatosis confined to the scalp. All the patients were elderly women who developed chronic, extensive, pustular, crusted and occasionally eroded lesions of the scalp which produced scarring alopecia. Investigations were essentially negative and skin biopsies showed only non-specific changes of atrophy and chronic inflammation, sometimes with increased plasma cells in the infiltrate. The condition did not respond to antibiotics, but was suppressed by potent topical steroids.


Male-pattern alopecia and masculinity.

Cutaneous processes which are thought to be influenced by androgenic stimulation include the development of male-pattern alopecia, terminal hair distribution, sebum excretion rate, maximal sweat secretion rate and skin thickness. We measured these indices in forty-eight normal men, together with muscle, fat and bone thickness and plasma testosterone. There was a significant correlation between hair density on the forearm, leg and chest, but no other significant correlations were found. We conclude that bald men are no more 'masculine' than those with good scalp hair growth, if masculinity is defined in terms of end-organ response to androgenic stimulation.


Diameter of the collagen fibrils in the sclerodermatous skin of porphyria cutanea tarda.

The diameter of collagen fibrils was studied in a patient with porphyria cutanea tarda in a specimen obtained from the sclerodermatous skin of the back. A bimodal distribution of the diameter of the fibrils was observed with maxima at 33.5 nm and 68.7 nm. The mean diameter was 67 nm with a standard deviation of 10.6 nm. These results are similar to those obtained previously in localized scleroderma (morphoea).


Scytalidium hyalinum infection of skin and nails.

Five cases of skin and nail infection associated with Scytalidium hyalinum are described. The lesions were confined to the hands, feet and toenails. All the patients were from the West Indies and were of Negroid descent. Scytalidium hyalinum was the only fungus isolated on repeated culture from three patients, while the remaining two patients had mixed infections, one with Trichophyton mentagrophytes var. interdigitale and the other with Hendersonula toruloidea.


A syndrome of ichthyosis, hepatosplenomegaly and cerebellar degeneration.

Two brothers are described with ichthyosis, hepatosplenomegaly and signs of cerebellar degeneration. The ichthyosis was similar to autosomal dominant ichthyosis both clinically and biochemically. No metabolic abnormality has yet been identified to account for this previously undescribed triad of abnormalities.


Dependence of acoustic attenuation of hearing protectors on incident sound level.

The relationship between incident sound level and acoustic attenuation for four types of earplug and four types of earmuff have been investigated using freshly prepared and instrumented cadaver ears. Pure tones and 1/3-octave bands of random noise in the frequency range 125-8000 Hz were employed as steady-state stimuli with sound pressure levels between 75 and 125 dB. Impulses with peak sound levels in the range 135-175 dB(P) were also presented. For the steady-state signals employed, the eight hearing protectors have been shown to have constant attenuation characteristics over the range of incident sound levels investigated. This was also the case for the six conventional protectors (with intentionally linear characteristics) for the impulse stimuli. The two intentionally amplitude-sensitive protectors provided attenuation which increased with incident sound level for impulse noises. Comparison of the protector attenuation-frequency characteristics determined for steady-state sounds shows good agreement with those obtained from subjective (threshold shift) national standard measurement procedures. It may be concluded, therefore, that the six conventional hearing protectors studied here have attenuation characteristics that are equal for incident sound levels at about 40 and 75 dB, and that they are constant for levels between 75 and 175 dB. Consequently, the results of national standard threshold-shift procedures, although measured at low sound levels, may be applied with confidence to occupations where hazardous high-level noises are present.


The mortality of men in the Rhondda Fach, 1950--1970.

A more detailed analysis of material from the 20-year follow-up of men in the Rhondda Fach confirms the similarity between the Standardised Mortality Ratios (SMRs) of miners and exminers with radiological categories 0, 1, 2, 3 and A (120.3, 116.5, 119.0, 115.7, and 120.1 respectively) as well as the difference between these SMRs and that of the non-moners (98.7). The specific death rates show a raised SMR for bronchitis and other respiratory diseases excluding pneumoconiosis for all categories including category 0, but little difference between those for category 0 and those for simple pneumoconiosis. The SMRs for ischaemic heart disease and other circulatory diseases for categories A, B and C combined are lower than those for simple pneumoconiosis and category 0 (84.2 and 85.0, compared with 109.8 and 121.8 for simple pneumoconiosis, and 117.5 and 114.6 for category 0). Fortunately the SMR for leukaemia is low. A comparison between the survival rates of men aged 55-64 in Leigh, Lancashire and those in the Rhondda Fach suggests that nonminers in the two areas have similar survival rates while the survival rates for category 0 and simple pneumoconiosis are lower in the Rhondda Fach.


Pulmonary function in firefighters: acute changes in ventilatory capacity and their correlates.

A group of 39 firefighters was examined during routine firefighing duty. Following smoke exposure the average decrease in one-second forced expiratory volume (FEV1.0) was 0.05 litre (137 observations). This decline in FEV1.0 was related to the severity of smoke exposure as estimated by the firefighter and to the measured particulate concentration of the smoke to which he was exposed. Decreases in FEV1.0 in excess of 0.10 litre were recorded in 30% of observations. Changes in FEV1.0 resulting from a second exposure to smoke on the same tour of duty were greater when smoke exposure at the previous fire was heavy. The repeated episodes of irritation of the bronchial tree that have been documented in this investigation may explain the origin of the previously observed chronic effect of firefighting on respiratory symptoms and pulmonary function.


Occupational lead exposure in Denmark: screening with the haematofluorometer.

The zinc protoporphyrin/haemoglobin (ZPP/Hb) ratio was measured in the field with a haematofluorometer. A significant increase in ZPP/Hb ratio with advancing age was found in 1295 men who denied any excess exposure to lead. Ninety-seven per cent of the results were below 110 mumol ZPP/mol Hb(Fe) (4.4 microgram ZPP/g Hb). The ZPP/Hb ratio was determined in a lead-exposed population of 2275 men, and in 305 a blood lead analysis was also performed. A blood lead limit of 2.9 mumol/l (60 microgram/100 ml) corresponds to about 500 mumol ZPP/mol Hb(Fe) (20 microgram/g). This limit was exceeded in workers engaged in secondary lead smelting, storage battery manufacture, car radiator repair, crystal glass manufacture, storage battery repair, ship breaking, metal foundries, the ceramic industry, scrap metal handling, and PVC plastic manufacture. Other occupations caused lower lead exposures with ZPP/Hb ratios between 110 and 500 mumol ZPP/mol Hb(Fe): such ratios were found in men from shooting ranges, in leaded pane manufacturers, gunsmiths, car paint sprayers, type setters, steel rolling mill workers, shipbuilders and welders, car mechanics, lead pigment handlers, and solderers. Increased ZPP/Hb ratios and blood lead levels in 210 workers were associated with a decrease in haemoglobin concentration in the blood. Thus, the haematofluorometer has proved to be very useful for screening purposes. A blood lead determination should be performed if the ZPP/Hb ratio exceeds 300 microgram ZPP/mol Hb(Fe) (12 microgram/g).


Bromine in blood, EEG and transaminases in methyl bromide workers.

In 33 methyl bromide users, slight electroencephalographic changes (in 10 subjects) and a small increase in serum transaminases were found which could be related to bromine concentration in blood. No relationship was found with subjective symptoms, general neurological examination or the results of serum protein electrophoresis.


Dimethylformamide and alcohol intolerance.

Facial flushing and other symptoms were reported by 19 of a group of 102 men who worked with dimethylformamide (DMF). Twenty-six of the 34 episodes occurred after the workers had consumed alcoholic drinks. The metabolite N-methylformamide (MF) was detected in the urine on 45 occasions, the highest recorded concentration being 77 microliter/litre. The highest recorded concentration of DMF in air was 200 ppm. The DMF-ethanol reaction is possibly attributable to the inhibition of acetaldehyde metabolism, probably by MF.


Counterion-induced condesation of deoxyribonucleic acid. a light-scattering study.

The addition of the trivalent or tetravalent cations spermidine or spermine to a solution of T7 DNA in aqueous solution causes an alteration of the DNA from its extended coil form to a condensed form. If performed at low DNA concentration and at low ionic strengths, this transformation results in a monomolecular collapse to form a particle with a hydrodynamic radius of about 500 A. We have monitored this change using quasielastic and total intensity light scattering. In a solution of 50% methanol in water, the divalent cations Mg2+ and putrescine also can cause the condensation of DNA. Using Manning's (1978) counterion condensation theory, we calculate a striking unity among these disparate ions: the collapse occurs in each case when from 89 to 90% of the DNA phosphate charges are neutralized by condensed counterions.


Chemical, physical-chemical, and immunological properties of papain-solubilized human transplatation antigens.

Papain-solubilized HLA-A, -B, and -C antigens have been isolated from cadaveric spleens. The isolated material was homogeneous and comprised subunits with the apparent molecular weights 33,000 and 12,000. Amino acid analyses of a mixture of HLA antigen heavy chains obtained from a great number of spleens with different HLA antigen phenotypes revealed a composition that is very similar to that of individual HLA-A and -B antigens. Likewise, the NH2-terminal 30 residues of the HLA-antigen heavy chain mixture were virtually identical with that recorded for individual specificities. The circular dichroism spectra for the isolated HLA antigens and for free beta2-microglobulin revealed similarities with spectra recorded for immunoglobulin chains and domains. The HLA-antigen heavy chain may contain an appreciable amount of beta structure. Antibodies raised against free beta2-microglobulin react better with beta2-microglobulin in free form than when bound to the HLA-A, -B, and -C antigen heavy chains. This is due to the fact that free beta2-microglobulin can bind a maximum of four Fab fragments simultaneously, whereas the HLA-antigen-associated beta2-microglobulin can bind only two Fab fragments without dissociating from the heavy HLA-antigen subunit.


Isolation, purification, and reconstitution of a proline carrier protein from Mycobacterium phlei.

Membrane vesicles from Mycobacterium phlei contain carrier proteins for proline, glutamine, and glutamic acid. The transport of proline is Na+ dependent and required substrate oxidation. A proline carrier protein was solubilized from the membrane vesicles by treatment with cholate and Triton X-100. Electron microscopic observation of the detergent-treated membrane vesicles showed that they are closed structures. The detergent-extracted proteins were purified by means of sucrose density gradient centrifugation, followed by gel filtration and isoelectric focusing. A single protein with a molecular weight of 20,000 +/- 1000 was found on polyacrylamide gel electrophoresis. Reconstitution of proline transport was demonstrated when the purified protein was incubated with the detergent-extracted membrane vesicles. This reconstituted transport system was specific for proline and required substrate oxidation and Na+. The purified protein was also incorporated into liposomes, and proline uptake was demonstrated when energy was supplied as a membrane potential introduced by K+ diffusion via valinomycin. The uptake of proline was Na+ dependent and was inhibited by uncoupler or by sulfhydryl reagents.


Structure of potato carboxypeptidase inhibitor: disulfide pairing and exposure of aromatic residues.

The determination of the covalent structure of a carboxypeptidase inhibitor from potatoes containing 39 amino acid residues has been completed by analysis of the pairing of the six half-cystine residues. Since the native inhibitor is resistant to fragmentation by proteases, the protein was first subjected to cleavage at aspartic acid residues by exposure to 0.03 N HCl at 110 degrees C for 10h to yield a fragment containing two chains (residues 6-15 and residues 18-39)held together by three disulfide bonds. Digestion with subtilisin and Pronase, respectively, yielded sets of peptides from which, by diagonal electrophoresis and amino acid analysis, the paired cystinyl residues were identified as Cys-8 to Cys-24, Cys-12 to Cys-27, and Cys-18 to Cys-34. Charge-transfer titration of the native inhibitor with N-methylnicotinamide chloride suggests that one of the two tryptophan residues and the single tyrosine residue are exposed to the solvent.


Lateral diffusion of M-13 coat protein in model membranes.

A fluorescent derivative of the M-13 phage coat protein (molecular weight 5260) was reconsituted into oriented multilayers and giant liposomes of dimyristoylphosphatidylcholine. The rate of lateral diffusion of the labeled protein in the fluid phase was measured as a function of temperature and found to be comparable to that of lipid probes. The protein was found to have a nonuniform lateral distribution in the solid phase of both types of model membranes. Cardiolipin (0.5 mol %) included in the multibilayers did not have any substantial effect upon the rate of diffusion.


Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme.

We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.


Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 2. Evidence for a substrate radical--enzyme complex and its reaction with nucleophiles.

Changes in the optical absorption spectrum of horseradish peroxidase, during the oxidation of o-dianisidine at pH 7.5, reveal an intermediate distinct from the previously described compounds I and II. The rate of decay of this new complex appeared to be rate limiting for the catalytic cycle, in this pH range, since imidazole, which augments the catalytic reaction, also enhanced the rate of decay of this complex. Nitrogenous compounds reportedly unable to ligate to hemes, such as 2-methylimidazole and benzimidazole, were nevertheless capable of augmenting the HRP-catalyzed rate of oxidation of o-dianisidine. The activity of nitrogenous compounds, in this regard, appeared to be a function of their nucleophilicity and was sensitive to steric factors but relatively free of a deuterium solvent isotope effect. The data presented in this and in the preceding paper [Claiborne, A., & Fridovich, I. (1979) Biochemistry 18 (preceding paper in this issue)] lead to the suggestion that the nucleophile-responsive intermediate is an enzyme--dianisidine radical complex and that abstraction of the second electron from the bound radical is facilitated by binding of nitrogenous nucleophiles.


Estrogen-binding proteins of calf uterus. Purification to homogeneity of receptor from cytosol by affinity chromatography.

The estrogen receptor has been purified to homogeneity from calf uterus cytosol by sequential affinity chromatography by using heparin--Sepharose 4B and 17-hemisuccinyl-17beta-estradiol-ovalbumin--Sepharose 4B. The procedure yields about 1.2 mg of receptor protein from 1 kg of calf uteri, with a recovery of 53%. The receptor protein, as a complex with 17beta-[3H]estradiol, is purified more than 99%. A single band is seen on polyacrylamide gel ectrophoresis under nondenaturing conditions. 17beta-[3H]Estradiol comigrates with the protein band. As computed from the specific activity of radioactive hormone, 64,450 g of purified receptor protein binds 1 mol of 17beta-estradiol. 17beta-[3H]Estradiol bound to the protein is displaced by estrogenic steriods but not by progesterone, testosterone, or cortisone. As judged by chromatography on calibrated Sephadex G-200 columns, the purified receptor is identical with native receptor in crude cytosol: both show a Stokes radius of 6.4 nm. On sucrose gradient in low-salt buffer, the purified receptor sediments at 8 S. On electrophoresis in NaDodSO4 gels, the purified receptor migrates as a single protein band with an apparent molecular weight of 70,000. The sedimentation coefficient measured on sucrose gradients in the presence of chaotropic salts [1 M NaBr or NaSCN (0.1 M)] is 4.2 S. We conclude that the estrogen receptor of cytosol consists of a single subunit weighing about 70,000 daltons and endowed with one estrogen binding site. Under native conditions in cytosol, several subunits associate to form a quaternary structure with a Stokes radius of 6.4 nm.


Effects of thio-group modification and Ca2+ on agonist-specific state transitions of a central nicotinic acetylcholine receptor.

Agonist-binding affinities of central nervous system nicotinic acetylcholine receptors (nAcChR) are sensitive to the duration of exposure to agonist. These agonist-induced changes in receptor state may be mimicked by appropriate modification of receptor thio groups and/or by manipulation of solvent ionic composition. In the absence of Ca2+, the concentration of acetylcholine (AcCh) necessary to prevent half of specific 3H-labeled alpha-bungarotoxin binding is approximately 1 mM for nAcChR treated with dithiothreitol (DTT) or DTT-N-ethylmaleimide (low-affinity states) and approximately 40 microM for nAcChR treated with DTT-5,5'-dithiobis(2-nitrobenzoic acid) or for native nAcChR pretreated with AcCh (high-affinity states). Addition of Ca2+ results in an increase in the effectiveness of AcCh toward blocking toxin binding. None of these treatments alters toxin or antagonist binding nor are there observed differences in Hill numbers for agonist binding. Agonists competitively inhibit toxin binding to low-affinity states, but noncompetitive inhibition is observed for binding to high-affinity states. Values of AcCh dissociation constants estimated from these data fall within the range of values determined physiologically with nAcChR from other systems. The data indicate that the redox state of brain nAcChR thio groups and Ca2+ may mediate physiologically important changes in the receptor state during activation and desensitization.


Relaxation kinetics of glutamate dehydrogenase self-association by pressure perturbation.

The kinetics of self-association for beef liver glutamate dehydrogenase (EC 1.4.1.3) have been measured by using pressure perturbation in both the time domain and the frequency domain by monitoring scattered light intensity. The kinetic behavior is entirely consistent with the random self-association model proposed by Thusius et al. [Thusius, D., Dessen, P., & Jallon, J. M. (1975) J. Mol. Biol. 92, 413--432]. The activation volume deltaV for association is estimated to be positive, and it is shown that this provides further corroboration of the molecular mechanism advanced by these same authors. A rapid shift in scattered light intensity is attributed to preferential interaction between the phosphate anion and the protein, proceeding with a positive volume change (2--5 mL/mol of phosphate). A description of the instrument developed for this study is also included.