Further studies on the effect of the collagen triple-helix formation on the hydroxylation of lysine and the glycosylations of hydroxylysine in chick-embryo tendon and cartilage cells.

The hydroxylation of lysine and glycosylations of hydroxylysine were studied in isolated chick-embryo tendon and cartilage cells under conditions in which collagen triple-helix formation was either inhibited or accelerated. The former situation was obtained by incubating the tendon cells with 0.6mm-dithiothreitol, thus decreasing their proline hydroxylase activity by about 99%. After labelling with [(14)C]proline, the formation of hydroxy[(14)C]proline was found to have declined by about 95%. Since the hydroxylation of a relatively large number of proline residues is required for triple-helix formation at 37 degrees C, the pro-alpha-chains synthesized under these conditions apparently cannot form triple-helical molecules. Labelling experiments with [(14)C]lysine indicated that the degree of hydroxylation of the lysine residues in the collagen synthesized was slightly increased and the degree of the glycosylations of the hydroxylysine residues more than doubled, the largest increase being in the content of glucosylgalactosylhydroxylysine. Recovery of chick-embryo cartilage cells from temporary anoxia was used to obtain accelerated triple-helix formation. A marked decrease was found in the extent of hydroxylation of the lysine residues in the collagen synthesized under these conditions, and an even larger decrease occurred in the glycosylations of the hydroxylysine residues. The results support the previous suggestion that the triple-helix formation of the pro-alpha-chains prevents further hydroxylation of lysine residues and glycosylations of hydroxylysine residues during collagen biosynthesis.


Studies on the metabolism of oestrone sulphate. Comparative perfusions of oestrone and oestrone sulphate through isolated rat livers.

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.


Sialyltransferase activity in regenerating rat liver.

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm-Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm-Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm-Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.


Structural requirements for the inhibitory action of 12,13-epoxytrichothecenes on protein synthesis in eukaryotes.

1. The inhibitory actions of ten trichothecene antibiotics were investigated, in reticulocyte cell-free systems synthesizing protein in vitro, by studying polyribosome profiles and kinetics of amino acid incorporation in the presence or absence of the drugs. 2. The modes of action observed were critically dependent on the drug concentrations used, but the antibiotics tested could be divided into four distinct groups, each exerting a characteristic inhibitory response. 3. The inhibitory action observed in every case was controlled by the chemical structure of the individual trichothecene and in particular was closely related to the nature of the substituent groups present on C-3, C-4, C-8 and C-15 of the molecule.


A rapid direct assay for the determination of the separate activities of the three arylsulphatases of Aspergillus oryzae.

1. The three arylsulphatases of Aspergillus oryzae exhibit pronounced kinetic differences and substrate specificities. Arylsulphatase I hydrolyses all substrates tested, whereas arylsulphatase III will not hydrolyse tyrosine O-sulphate or phenolphthalein disulphate. Arylsulphatase II does not hydrolyse p-nitrophenyl sulphate or phenolphthalein disulphate at appreciable rates in the absence of added phenolic compounds. Phenols such as tyramine increase the rate of hydrolysis of these substances by this enzyme 1000-fold. At pH 6.9 arylsulphatase I exhibits an apparent Km of 0.1 mM for p-nitrophenyl sulphate, whereas the Km of arylsulphatase III for this substrate is 1 mM. 2. These differences were utilized to develop an assay procedure which can be used to determine the separate activities of the three enzymes present in mixtures. This assay has potential use as a means of examining the relative activities of the three enzymes in investigations of the differences in the mechanisms regulating their synthesis.


Differential repression of arylsulphatase synthesis in Aspergillus oryzae.

1. The activities of the three arylsulphatases (arylsulphate sulphohydrolase, EC 3.1.6.1) of Aspergillus oryzae produced under a variety of repressing and non-repressing conditions were determined. 2. These enzymes exhibit different sensitivities to repression by inorganic sulphate. 3. Arylsulphatase I, but not arylsulphatases II and III, exhibits a transient de-repression in the early growth phase in sulphate media. 4. When the fungus is cultured in repressing media and subsequently transferred to non-repressing media, the synthesis of the three enzymes is non-co-ordinate. 5. Growth of the fungus in media containing choline O-sulphate or tyrosine O-sulphate as the sole source of sulphur results in complete de-repression of arylsulphatase I, But the synthesis of arylsulphatases II and III is essentially fully repressed. 6. The marked similarities between the repression characteristics of arylsulphatases II and III, contrasted with those of arylsulphatase I, indicate that the genetic locus of arylsulphatase I is distinct from that of arylsulphatases II and III, suggesting that there are distinct physiological roles for the enzyme.


The association of the sulphogalactosylglycerolipid of rat brain with myelination.

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4mumol per brain) thereafter into adult life. 2. The activity of adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H(2) (35)SO(4) at 20 days of age lost (35)S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.


The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro.

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.


Synthesis of procollagen by matrix-free cells from embryonic-chick arteries.

Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [(14)C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[(14)C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-alpha-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.


Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

1. The metabolism of protein and phospholipid in rat liver plasma membranes isolated by the method of Neville [(1960) J. Biophys. Biochem. Cytol. 8, 413-422] was investigated 3 and 6 h after the injection of D-galactosamine in vivo. During this time, all the biochemical and morphological alterations associated with hepatitis developed. 2. After the injection of D-galactosamine the concentration of sphingomyelin in the plasma membrane decreased to below 60% of the control values. 3. The activity of 5'-nucleotidase (EC 3.1.3.5), which has been purified as a sphingomyelin-protein complex, decreased in the total homogenate as well as in the plasma-membrane fraction of livers of rats treated with galactosamine, to about 60% of the control values. 4. Protein synthesis, as measured by the incorporation of [14C]leucine into plasma membranes, was decreased to 45% of that of the controls. However, only small differences were observed in the amino acid composition of the plasma membrane after D-galactosamine treatment. 5. The protein composition of the plasma membranes was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results showed a change from low- to high-molecular-weight proteins after the injection of galactosamine. 6. These results demonstrate different metabolic processes of the plasma membrane altered during the induction of galactosamine hepatitis.


Metabolism and transport of glutamine and glucose in vascularly perfused small intestine rat.

1. The metabolism and transport of glutamine and glucose were investigated in a preparation of rat small intestine perfused through the vascular bed in vitro and in situ. 2. With glucose (7.5mm) or glutamine (4.5mm) in the lumen, approx. 40% of the substrate taken up appears unchanged on the vascular side. When glutamine (1.5mm) is also added to the vascular perfusate, metabolism of glutamine is increased and there is uptake of glutamine from both the vascular bed and lumen. Orientation of substrate (vascular bed or lumen) influences the value of alanine production/glutamine utilization and lactate production/glucose utilization. 3. Deprivation of food and metabolic acidosis have no effect upon the utilization of glutamine by unit length of jejunum. In fed rats, glutamine utilization is 44% of glucose utilization, but in rats deprived of food it is 112% of glucose utilization. 4. Glucose utilization and lactate production are not significantly altered by the presence of glutamine in the vascular bed or lumen. 5. With glucose only in the vascular perfusate, glucose utilization is the same in jejunum and ileum. Glutamine metabolism in the ileum is 28% lower than in the jejunum. 6. Glutamine utilization is dependent on the concentration of glutamine in the vascular perfusate, but is not significantly affected by the absence of glucose. 7. Results are discussed in relation to the role of intestinal glutamine metabolism and with respect to some problems of the transepithelial movement of substrates that are both transported and metabolized.


Tissue distribution of S-adenosylmethionine and S-adenosylhomocysteine in the rat. Effect of age, sex and methionine administration on the metabolism of S-adenosylmethionine, S-adenosylhomocysteine and polyamines.

The tissue distribution of S-adenosylmethionine, S-adenosylhomocysteine, methionine adenosyltransferase and S-adenosylhomocysteine hydrolase was explored in the rat. Also the effects of methionine administration on the accumulation of S-adenosylmethionine, S-adenosylhomocysteine and polyamines were studied in rat liver, brain and kidney. The tissue distribution of S-adenosylmethionine, S-adenosylhomocysteine, methionine adenosyltransferase and S-adenosylhomocysteine hydrolase was similar in both sexes, and was only slightly changed with age. The specific activity of S-adenosylhomocysteine hydrolase greatly exceeded that of methionine adenosyltransferase, and the concentration of S-adenosylmethionine was higher than that of S-adenosylhomocysteine in all tissues examined. However, the hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio was dependent on food supply and on the age of the animal. No correlation was noticed between the activity of methionine adenosyltransferase and the concentrations of the adenosyl compounds in different tissues. Intraperitoneal administration of methionine resulted in a profound but transient increase in the hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine. The concentration of S-adenosylmethionine was elevated also in the brain during the first 2h after methionine injection. The rise of S-adenosylmethionine concentration after methionine treatment could be diminished by simultaneous glycine administration. The results support the view that the rate-limiting factor of S-adenosylmethionine synthesis is the tissue concentration of methionine. They further suggest that glycine N-methyltransferase may have a regulatory role in the utilization of S-adenosylmethionine in the liver.


The effect of calcium on lead absorption in rats.

The effects of Ca2+ on lead absorption as PbCl2 and 203PbCl2 were studied in rats. 1. Doubling of dietary calcium with Ca3(PO4)2 significantly decreased lead absorption as assessed by 203Pb retention, tissue lead concentration, urinary excretion of delta-aminolaevulinate and increased activities of delta-aminolaevulinate dehydratase and ferrochelatase. 2. Similar effects on lead absorption were shown by the Ca2+ salts, Cl-, CO32-, PO43-, SO42-, gluconate and glycerophosphate. 3. CaCl2 and calcium glycerophosphate were found to be most effective in decreasing lead absorption when administered immediately before lead dosage. 4. A negative exponential relationship was found between CaCl2 concentration and 203Pb absorption at 120h. The result suggest that, above 4 mmol of administered calcium, residual lead absorption is unaffected by increasing gastrointestinal calcium concentrations. 5. Increased systemic calcium had no effect on lead retention. 6. Calcium in the concentrations found in domestic hard-water supplies significantly decreased absorption of a solution of 203Pb dissolved in it compared with absorption of 203Pb dissolved in soft or distilled water. 7. Milk and skimmed milk were found to have no effect on 203Pb absorption in rats.


Origins of blood acetate in the rat.

A novel enzymimc cycling assay for the determination of acetate in biological material is described. Measurements of the acetate concentration in blood and liver samples from rats of various ages and nutritional states with this assay are reported. The contribution of the intestine, the liver and the rest of the body to maintaining the concentration of acetate in the circulation is examined. Evidence is presented that the gut flora constitute the main source of acetate in blood of fed adult rats, though endogenous production of acetate is of significance in other situations. The streptozotocin-diabetic rat has an elevated blood acetate concentration.


The effects of inhibition of fatty acid oxidation in suckling newborn rats.

Inhibition of fatty acid oxidation with pent-4-enoate in suckling newborn rats caused a fall in blood [glucose] and blood [ketone bodies] and inhibition of gluconeogenesis from lactate. Glucose utilization was not increased in newborn rats injected with pent-4-enoate. Active fatty acid oxidation appears to be essential to support gluconeogenesis and to maintain normal blood [glucose] in suckling newborn rats.


The effects of acute ethanol feeding and of chronic benfluorex administration on the activities of some enzymes of glycerolipid synthesis in rat liver and adipose tissue.

Rats were treated for 5 days with benfluorex [1-(3-trifluoromethylphenyl)-2-[N-(2-benzoyloxyethyl)amino]propane] or with suspending medium (controls). They were then intubated with an acute intoxicating dose of ethanol or with glucose of equivalent energy content. Treatment of the control rats with ethanol specifically increases the hepatic activity of the soluble phosphatidate phosphohydrolase by about 5-fold in 6 h. The equivalent increase for the benfluorex-treated rats were about 2-fold. The results are discussed in relation to the effects of ethanol and benfluorex on glycerolipid synthesis.


Oestrogen induction of riboflavin-binding protein in immature chicks.

In hyperthyroid chicks, oestrogen-induced plasma accumulation of riboflavin-binding protein was diminished, whereas the reverse situation prevailed in hypothyroid birds. Under hyperthyroid conditions, higher concentrations of oestrogen were required to elicit a response comparable with that obtained in normal birds treated with lower concentrations of the hormone. Elevated hepatic cytochrome P-450 concentrations and decreased half-life of the induced protein in hyperthyroid animals suggest that higher catabolic rates of the inducer and induced protein are contributory factors to the diminished response.


Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex.

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.


A subunit-sized butyrylcholinesterase present in high concentrations in pooled rabbit serum.

A butyrylcholinesterase of mol.wt. approx. 83000 was observed in pooled rabbit serum. The enzyme was named monomeric butyrylcholinesterase to distinguish it from the larger oligomeric butyrylcholinesterase of horse and human serum whose subunits are the same size as the monomeric enzyme. The active-site concentration of monomeric butyrylcholinesterase in the pooled serum was 0.18mum, which is five times the concentration of butyrylcholinesterase in pooled horse serum. This was surprising, since the horse serum is regarded as a rich source of butyrylcholinesterase, whereas rabbit serum is not generally thought to contain significant amounts of any butyrylcholinesterase. The explanation, in large part, was the relatively low k(cat.) of the monomeric enzyme, which was approx. 57s(-1) with butyrylthiocholine as substrate and is one-thirtieth of the comparable k(cat.) of horse butyrylcholinesterase. The substrate specificity of monomeric butyrylcholinesterase also differed significantly from that of horse and human butyrylcholinesterase. For example, with the monomeric enzyme, the hydrolysis of 1mm-acetylthiocholine was only 4% the rate for 1mm-butyrylthiocholine, whereas human and horse butyrylcholinesterases hydrolysed 1mm-acetylthiocholine at 50% of the rate for 1mm-butyrylthiocholine. Moreover, monomeric butyrylcholinesterase generally hydrolysed aromatic esters more rapidly than choline esters, whereas the reverse is true of the butyrylcholinesterases. To facilitate the study of monomeric butyrylcholinesterase, it was separated from the larger butyrylcholinesterase and acetylcholinesterase, also present in rabbit serum, and purified 89-fold by fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography.


Studies on C3 convertases. Inhibition of C5 convertase formation by peptides containing aromatic amino acids.

The influence of various peptides containing the aromatic amino acids phenylalanine and tyrosine on the formation of the enzyme EAC1423 of the complement system from component C3 and enzyme EAC142 was investigated. Kinetic analysis of enzyme EAC1423 formation and studies on the binding of the C3b fragment of 125I-labelled component C3 to enzyme EAC142 both showed that binding of the C3b fragment of component C3 was decreased by the peptides. Kinetic studies on component-C3 turnover in the fluid phase of enzyme EAC142 failed to reveal effects of the peptides. However, an initial lag in component-C3 turnover occurred that at constant component-C3 concentration was inversely proportional to enzyme EAC142 concentration. This lag in enzyme EAC142 activity is considered as an indication that the interaction of enzyme EAC142 with component C3 possibly does not follow simple Michaelis-Menten kinetics, as was previously assumed. It is shown that the stages after enzyme EAC1423 formation are not influenced by the peptides, suggesting a high degree of specificity of the peptides for the inhibition of enzyme EAC1423 formation.


Affinity chromatography of aminoacyl-transfer ribonucleic acid synthetases. Small organic ligands.

The usefulness of affinity chromatography for the purification of aminoacyl-tRNA synthetases was explored by using column ligands derived from the corresponding amino acid and aminoalkyladenylate, a non-labile analogue of the aminoacyladenylate reaction intermediate. Four modes of attachment of the aminoalkyladenylate to Sepharose were studied. The interaction between amino acid derivatives and the corresponding aminoacyl-tRNA synthetases is too weak to allow their use as ligands for affinity chromatography. Attachment of the aminoalkyladenylate via the alpha-nitrogen atom of the amino acid or via C-8 of the nucleotide abolishes synthetase binding, and immobilization via the oxidized ribose ring is only marginally useful. However, attachment of the aminoalkyladenylate to the matrix via N-6 of the nucleotide allows strong and specific synthetase binding, and the use of such columns permits the isolation of homogeneous synthetase from crude mixtures. The effect of non-specific adsorption and the utility of pre-columns and of specific substrate elution are investigated and discussed.


A quantitative analysis of histone H1 in rabbit thymus nuclei.

The relative quantity of histone H1 in rabbit thymus whole histone was determined to be 17.2% (w/w). This implies that there is, on average, one histone H1 molecule per nucleosome.


A cystine-rich protein fraction from oxidized alpha-keratin.

A soluble fraction of alpha-keratin was obtained on fission of disulphide bonds. The fraction was soluble in the oxidizing solution and would normally be lost when such procedures are used for isolating keratose fractions. This fraction, which constituted 6% by weight of keratin, was rich in cystine, and about 30% of the fraction had a mol.wt. of less than 20 000.


Electrochemical analysis of the carboxy-14C-labelled aliphatic carboxylic acid metabolites resulting from tracer studies.

14C02 output from carboxy-14C-labelled aliphatic carboxylic acids is measured in the micro-scale Kolbe reaction. Irrespective of whether rats were dosed with 1,1-dichloro[1-14C]ethylene or with chloro[1-14C]acetic acid, 1 mol.equiv. of the resulting thio[14C]diglycollic acid yields by electrolysis approx. 0.7 equiv. of 14CO2, which is interpreted in terms of the labelling of one of the carboxylic acid groups of thiodiglycollic acid. This observation provides important evidence concerning thiodiglycollic acid biosynthesis from 1.1-dichloroethylene.


The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans.

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.


Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes.

Free loosely bound and tightly bound polyribosomes were separated from rat liver homogenate by salt extraction followed by differential centrifugation, and several of their structural and functional properties were compared to resolve the existence of loosely bound polyribosomes and verify the specificity of the separation. The free and loosely bound polyribosomes have similar sedimentation profiles and polyribosome contents, their subunit proteins have similar electrophoretic patterns and their products of protein synthesis in vitro show a close correspondence in size and amounts synthesized. In contrast, the tightly bound polyribosomes have different properties from those of the free and loosely bound polyribosomes; their average size is significantly smaller; their polyribosome content is higher; their 60 S-subunit proteins lack two components and contain four or more components not found elsewhere; their products of protein synthesis in vitro differ in size and amounts synthesized. These observations show that rat liver membranes entrap a large fraction of the free polyribosomes at low salt concentrations and that these polyribosomes are similar to those of the free-polyribosome fraction and are different from those of the tightly bound polyribosome fraction in size, structure and function.


Accelerated hepatic haem catabolism in the selenium-deficient rat.

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.


The cytochromes of Acanthamoeba castellanii.

1. Low-temperature difference spectra of gradient-purified mitochondria of Acanthamoeba castellanii reveal the presence of cytochromes b-555, b-562 and c-549, with a-type cytochromes having a broad asymmetrical maximum at 602 nm; these components were also observed in specta of whole cells. 2. The a-type cytochromes are unusual in that they have split Soret absorption maxima (at 442 and 449 nm) and an uncharacteristic CO difference spectrum. 3. CO difference spectra of whole cells and 'microsomal' membranes show large amounts of cytochrome P-420 compared with cytochrome P-450. 4. Difference spectra in the presence of cyanide indicate the presence of an a-type cytochrome and two cyanide-reacting components, one of which may be cytochrome a3. 5. Whole-cell respiration in a N2/O2 (19:1) atmosphere was decreased by 50%, suggesting the presence of a low-affinity oxidase. This lowered respiration is inhibited by 50% by CO, and the inhibition is partially light-reversible; photochemical action spectra suggest that cytochrome a3 contributes to this release of inhibition. Other CO-reacting oxidases are also present. 6. The results are discussed with the view that cytochrome a3 is present in A. castellanii, but its identification in CO difference spectra is obscured by other component(s).


Energy-dependent uptake of arsenite by rat liver mitochondria.

Uptake of arsenite by rat liver mitochondria is energy-dependent, as shown by comparing values without and with either uncoupling agent or respiratory inhibitor present. The uptake is inhibited by mersalyl and N-ethylmaleimide, which can be used as 'stopping' agents to obtain uptake kinetics. At 20 degrees C the process is nearly complete in 1 min. The relation between the quantity in the energized mitochondria and the applied concentration corresponds to at least two different modes of binding of the arsenite. Competition occurs between arsenite and other anions (for example, phosphate) for intramitochondrial accumulation.


Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis.

The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [(3)H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [(3)H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [(3)H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis, which resembles RNA synthesis in spermatocytes in vivo. Therefore isolated spermatocytes of the rat can be used for studying the possible regulation of RNA synthesis during the meiotic prophase.


Hormonal control of gluconeogenesis in tubule fragments from renal cortex of fed rats. Effects of alpha-adrenergic stimuli, glucagon, theophylline and papaverine.

1. In incubated tubule fragments from renal cortex of fed rats gluconeogenesis from pyruvate was stimulated by adrenaline (1mum optimum) and by the selective alpha-adrenergic agonists oxymetazoline and amidephrine. The selective beta-agonists isoproterenol and salbutamol were ineffective at concentrations up to 10mum. 2. Stimulation of gluconeogenesis by 1mum-adrenaline was almost completely blocked by 10mum-phentolamine (alpha-antagonist), partially blocked by 10mum-phenoxybenzamine (alpha-antagonist) and unaffected by 10mum-propranolol (beta-antagonist). 3. Adrenaline stimulation of gluconeogenesis was rapid and was sustained for at least 1h. 4. Oxymetazoline (alpha-agonist) was extremely potent in stimulation of gluconeogenesis. This compound stimulated glucose production from pyruvate, lactate and glutamate, but not from succinate or glycerol. 5. In the absence of Ca(2+) oxymetazoline was ineffective, whereas some stimulatory effect of adrenaline on gluconeogenesis was still observed. 6. Glucagon had no effect on gluconeogenesis from pyruvate in the presence of 1.27mm-Ca(2+) and inhibited the process in the presence of 0.25mm-Ca(2+). Parathyrin (parathyroid hormone) stimulated gluconeogenesis at 1.27mm-Ca(2+). 7. In short incubations of tubule fragments glucagon, papaverine and adrenaline significantly increased 3':5'-cyclic AMP. Adrenaline also slightly decreased 3':5'-cyclic GMP. Oxymetazoline had no effect on the amount of either cyclic nucleotide. 8. At all concentrations tested, theophylline and papaverine decreased gluconeogenesis from pyruvate. 9. It is concluded that renal gluconeogenesis may be increased by alpha- but not beta-adrenergic stimuli and that this is probably independent of changes in 3':5'-cyclic AMP or 3':5'-cyclic GMP. An involvement of Ca(2+) in the action of oxymetazoline appears likely, but this is less certain with adrenaline.


Ornithine decarboxylase and polyamines in liver and kidneys of rats on cyclical regimen of protein-free and protein-containing diets. Relationship to deoxyribonucleic acid synthesis in liver.

1. The activity of ornithine decarboxylase in the liver and kidneys of rats maintained on a cyclical regimen of protein-free and protein-containing diets was investigated. There was a daily activation of the enzyme in response to the feeding of protein after 3 days feeding of protein-free diet. 2. The activation of ornithine decarboxylase in the liver and kidneys of rats re-fed on protein was demonstrable throughout 16 cycles of alternating 3-day periods of protein-free and protein-containing diets. The magnitude of the activation in the kidneys diminished from 20-fold stimulation in the first cycle to 5-fold stimulation (compared with animals fed with protein-free diet) in the later cycles of protein re-feeding. The activation of the enzyme in liver was decreased from 20-fold stimulation in the first cycle to approx. 10-fold stimulation in later cycles. 3. The concentration of spermidine was increased by approx. 50% in the liver of animals during cycling from protein-free to protein-containing diets. Spermine was unchanged, and putrescine was maintained at a low concentration approx. one-fifth to one-tenth that of spermidine after protein re-feeding. 4. The incorporation of [(3)H]thymidine into liver DNA was increased 10-fold in animals re-fed with protein compared with animals receiving protein-free diets. 5. The activation of ornithine decarboxylase by re-feeding of protein was inhibited 90% by the injection of propane-1,3-diamine during re-feeding. The stimulation of DNA synthesis was inhibited 60% by multiple injections of propane-1,3-diamine during the re-feeding of protein.


Regulation of mammalian protein synthesis in vivo. Stimulated liver ribonucleic acid synthesis in vivo after cycloheximide treatment.

1. As shown by a double-radioisotope technique in vivo, at a non-lethal dose of cycloheximide, a stimulation of nuclear RNA synthesis occurred by 12 h after the treatment; the stimulation lasted over 48 h. Analysis of radioactive nuclear RNA by gel electrophoresis demonstrated that most of the cycloheximide-stimulated synthesis could be accounted for by known rRNA precursors (45 S, 41 S, 32 S and 28 S). 2. During the inhibitory phase of protein synthesis, 2 h after cycloheximide treatment, synthesis of the poly(A)-containing mRNA isolated from the cytoplasmic ribonucleoprotein complexes with an oligo(dT)-cellulose column was stimulated, whereas the synthesis of rRNA was slightly inhibited. However, during the stimulatory phase of protein synthesis, 24 h after cycloheximide treatment, the syntheses of both poly(A)-containing mRNA and rRNA were enhanced. 3. Kinetic studies revealed that the newly synthesized RNA species were transported from the nuclei, integrated into the ribonucleoprotein complexes, and associated with both free and membrane-bound polyribosomes. 4. These data corroborate our proposal that the stimulated protein synthesis after cycloheximide administration involves gene transcription.


The mode of action of 4-methylumbelliferyl beta-D-xyloside on the synthesis of chondroitin sulphate in embryonic-chicken sternum.

1. Embryonic-chicken sterna, incubated in medium containing 0.1mm-4-methylumbelliferyl beta-d-xyloside (4-methylcoumarin 7-beta-d-xyloside), synthesize proteochondroitin sulphate that is significantly undersulphated and shorter than usual [Gibson, Segen & Audhya (1977) Biochem. J.162, 217-233]. 2. Neither the beta-d-galactoside nor the beta-d-glucuronide of 4-methylumbelliferone, nor 4-methylumbelliferone itself, produced the effects. The only metabolites of 4-methylumbelliferone that were detected in cartilages exposed to 4-methylumbelliferyl beta-d-xyloside were unchanged xyloside and chondroitin sulphate covalently attached to 4-methylumbelliferone. 3. Gel filtration of salt extracts of sterna incubated in medium containing the xyloside showed that there were two pools of chondroitin sulphate in the tissue. One pool was identified, on the basis of its elution pattern and the linear kinetics of incorporation of sulphate into it, as proteochondroitin sulphate. Incorporation into the other pool, whose properties suggested that it was methylumbelliferyl-chondroitin sulphate, indicated that it underwent partial turnover. The molecular weight of this chondroitin sulphate was about 19000, and it appeared to be about 70% sulphated. 4. When sterna were incubated in medium containing the xyloside, there was a very large incorporation of sulphate and glucose into glycosaminoglycans that were released into the incubation medium. This contrasts with incubations of sterna in the absence of the xyloside, in which less than 5% of the sulphate incorporated could be recovered from the medium. The glycosaminoglycan released into the medium was 4-methylumbelliferyl-chondroitin sulphate, whose average molecular weight was 7000-8000 and degree of sulphation more than 95%. 5. Incorporation of sulphate into proteochondroitin sulphate was stimulated more than 3-fold by addition of 20% (v/v) human serum and 10nm-l-3,3',5-tri-iodothyronine. Incorporation into methylumbelliferyl-chondroitin sulphate, in either the tissue or the medium, was not significantly altered. 6. The decrease in chain length and degree of sulphation of proteochondroitin sulphate is explained in terms of competition between peptide-linked primers and methylumbelliferone-containing primers at the intracellular sites of polysaccharidechain elongation and sulphation. The implications of the results for the mechanism of stimulation of proteoglycan synthesis by serum factors are discussed.


Immunological evaluation of repeated administration of erythrocyte-entrapped protein to C3H/HeJ mice.

The immunogenicity of heterologous protein entrapped in autologous erythrocytes was evaluated in a mouse system. Multiple intravenous administrations of an erythrocyte-entrapped bovine beta-glucuronidase preparation resulted in little, if any, immunological response to the entrapped protein or erythrocyte carrier. Erythrocyte entrapment also protected protein from circulating antibody elicited by prior subcutaneous sensitization with unentrapped enzyme in adjuvant. Thus erythrocyte entrapment may protect potentially immunogenic agents from immunological surveillance.


Iodinated phospholipids and the in vitro iodination of proteins of dog thyroid gland.

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.


Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle.

The effects of exogenous oleate on glucose uptake, lactate production and glycogen concentration in resting and contracting skeletal muscle were studied in the perfused rat hindquarter. In preliminary studies with aged erythrocytes at a haemoglobin concentration of 8g/100ml in the perfusion medium, 1.8mm-oleate had no effect on glucose uptake or lactate production. During these studies it became evident that O(2) delivery was inadequate with aged erythrocytes. Perfusion with rejuvenated human erythrocytes at a haemoglobin concentration of 12g/100ml resulted in a 2-fold higher O(2) uptake at rest and a 4-fold higher O(2) uptake during muscle contraction than was obtained with aged erythrocytes. Rejuvenated erythrocytes were therefore used in subsequent experiments. Glucose uptake and lactate production by the well-oxygenated hindquarter were inhibited by one-third, both at rest and during muscle contraction, when 1.8mm-oleate was added to the perfusion medium. Addition of oleate also significantly protected against glycogen depletion in the fast-twitch red and slow-twitch red types of muscle, but not in white muscle, during sciatic-nerve stimulation. In the absence of added oleate, glucose was confined to the extracellular space in resting muscle. Addition of oleate resulted in intracellular glucose accumulation in red muscle. Contractile activity resulted in accumulation of intracellular glucose in all three muscle types, and this effect was significantly augmented in the red types of muscle by perfusion with oleate. The concentrations of citrate and glucose 6-phosphate were also increased in red muscle perfused with oleate. We conclude that, as in the heart, availability of fatty acids has an inhibitory effect on glucose uptake and glycogen utilization in well-oxygenated red skeletal muscle.


S-adenosylmethionine metabolism and its relation to polyamine synthesis in rat liver. Effect of nutritional state, adrenal function, some drugs and partial hepatectomy.

S-Adenosylmethionine metabolism and its relation to the synthesis and accumulation of polyamines was studied in rat liver under various nutritional conditions, in adrenalectomized or partially hepatectomized animals and after treatment with cortisol, thioacetamide or methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}. Starvation for 2 days only slightly affected S-adenosylmethionine metabolism. The ratio of spermidine/spermine decreased markedly, but the concentration of total polyamines did not change significantly. The activity of S-adenosylmethionine decarboxylase initially decreased and then increased during prolonged starvation. This increase was dependent on intact adrenals. Re-feeding of starved animals caused a rapid but transient stimulation of polyamine synthesis and also increased the concentrations of S-adenosylmethionine and S-adenosylhomocysteine. Similarly, cortisol treatment enhanced the synthesis of polyamines, S-adenosylmethionine and S-adenosylhomocysteine. Feeding with a methionine-deficient diet for 7-14 days profoundly increased the concentration of spermidine, whereas the concentrations of total polyamines and of S-adenosylmethionine showed no significant changes. The results show that nutritional state and adrenal function play a significant role in the regulation of hepatic metabolism of S-adenosylmethionine and polyamines. They further indicate that under a variety of physiological and experimental conditions the concentrations of S-adenosylmethionine and of total polyamines remain fairly constant and that changes in polyamine metabolism are not primarily connected with changes in the accumulation of S-adenosylmethionine or S-adenosylhomocysteine.


Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway.

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2--3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.


Skeletal-muscle sarcolemma from normal and dystrophic mice. Isolation, characterization and lipid composition.

1. Mouse skeletal-muscle sarcolemma was isolated, and the preparations obtained from normal mouse muscle and from muscle of mice with hereditary muscular dystrophy were characterized with respect to appearance under the optical and electron microscopes, distribution of marker enzymes, histochemical properties and biochemical composition. 2. The sarcolemmal membranes from normal and dystrophic muscle were subjected to detailed lipied analysis. Total lipid content was shown to increase in sarcolemma from dystrophic mice as a result of a large increase in neutral lipid and a smaller increase in total phospholipids. Further analysis of the neutral-lipid fraction showed that total acylglycerols increased 6-fold, non-esterified fatty acid 4-fold and cholesterol esters 2-fold, whereas the amount of free cholesterol remained unchanged in sarcolemma from dystrophic muscle. Significant increases were found in lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in dystrophic-muscle sarcolemma; however, the relative composition of the phospholipid fraction remained essentially the same as in the normal case. 3. The overall result of alterations in lipid composition of the sarcolemma in mouse muscular dystrophy was an increase in neutral lipid compared with total phospholipid, and a 4-fold decrease in the relative amount of free cholesterol in the membrane. The possible impact of these changes on membrane function is discussed.


The pinocytosis of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by rat yolk sac cultured in vitro.

The rates of uptake of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by 17.5-day rat yolk sac cultured in vitro were studied. Over a 6.5h period each substrate was accumulated at a constant and reproducible rate of approx. 2microliter/h per mg of protein. After accumulation in vitro, the three substances were released from the tissue into substrate-free medium at low rates. Sucrose present in the medium at concentrations up to 10 mg/ml was without effect on the accumulation of either [14C]sucrose or 125I-labelled poly(vinylpyrrolidone), but at higher concentrations inhibited the uptake of both substrates. Some batches of colloidal [198Au]gold had a significantly higher Endocytic Index (up to 5 microliter/h per mg of protein). The Endocytic Index of such a batch decreased with increasing substrate concentration, but colloidal gold did not decrease the Endocytic Index of 125I-labelled poly(vinylpyrrolidone). It is concluded that the three substrates enter the yolk sac by pinocytosis in the liquid phase. Those batches of colloidal [198Au]gold with higher Endocytic Indices are considered to enter also by adsorption on membrane binding-sites.


The inhibition of lipid autoxidation by human caeruloplasmin.

1. Purified caeruloplasmin was shown to inhibit lipid autoxidation induced by ascorbic acid or inorganic iron in the following systems: (a) an emulsion of linolenic acid in water; (b) an untreated ox brain homogenate in phosphate buffer; (c) a similar homogenate whose susceptibility to autoxidation had been abolished by dialysis or by heating and then restored by the above pro-oxidants. 2. The optimum conditions for this antioxidant activity were studied. 3. Caeruloplasmin did not inhibit autoxidation by u.v. irradiation in dialysed or preheated homogenates. 4. The apoprotein (without copper) had no antioxidant activity, whereas CuSO4 alone was much less effective as an antioxidant. 5. Iron-free transferrin also had some antioxidant activity.


Incorporation of D-glucose-, L-alanine- and phosphate-transport systems from rat renal brush-border membranes into liposomes.

An extract of soluble proteins was prepared from a rat kidney brush-border membranes by Triton X-100 solubilization followed by centrifugation for 1 h at 100000g. Its protein composition was markedly different from that of the brush-border membranes. Proteoliposomes were formed by co-sonication of the Triton X-100-free extract with a naturally occurring mixture of phospholipids extracted from rat kidney. These proteoliposomes were shown to contain Na+-stimulated D-glucose-, L-alanine- and phosphate-transport systems.


alpha-Pinene metabolism by Pseudomonas putida.

By using metabolically altered mutants and acrylate, novel putative intermediates of alpha-pinene metabolism by Pseudomonas putida PIN11 were detected. They were characterized as 3-isopropylbut-3-enoic acid and (zeta)-2-methyl-5-isopropylhexa-2,5-dienoic acid.


A comparison of the effects of prednisolone and methylprednisolone on human lymphoblastoid cells.

Methylprednisolone is more effective than prednisolone in mediating long-term cytolysis and immediate inhibition of nucleoside uptake in human lymphoblastoid cells. This suggests that methylation of prednisolone makes it more potent by aiding interaction with a cellular target rather than increasing its stability.


Metabolic intermediates, enzymes and lysosomal activity in aortas of spontaneously hypertensive rats.

Metabolic intermediate levels, glycolytic and Krebs cycle enzyme activities and lysosomal acid hydrolase activities were measured in aortas of spontaneously hypertensive (SHR) versus normotensive (WKY) rats. In the hypertensive aortas the level of lactate, the ratio of lactate to glucose and of lactate to malate was higher in the SHR than WKY aortas. In the hypertensive aortas the obvious shift of metabolism toward higher rate of glycolysis was associated with decreased activity of malate dehydrogenase and espically of lipoamide dehydrogenase. The latter is an essential compoenent of the alpha-ketoglutarate and pyruvate dehydrogenase enzyme complexes and it appears that these complexes are among the sites of arterialmetavolism which are primarily altered by the elevated blood pressure, resulting in increased production of lactate. The activity of the marker lysosomal enzyme N-acetyl-beta-glucosaminidase was unequivocally elevated in the hypertensive aortas. The activity of beta-glucuronidase exhibited incogruous differences between the SHR and WKY aortas and the activity of aortic acid phosphatase did not differ in the two rat strains. The results are discussed in relation to arterial injury, permeability, and atherogenesis.


Role of complement in thrombogenesis. I. Complement-dependent coagulation in a model system.

Chondroitin sulphate was used to isolate from plasma a system that clotted with Russell's viper venom, brain extract and activated contact factors. Clotting appeared to depend on concomitant change in C4, C3 and C1s in the system. Brain extract additionally reacted with C9. Reconstitution of specifically defective plasmas suggested a specific role for each of these complement components in clotting.


Plant sterols as cholesterol-lowering agents: clinical trials in patients with hypercholesterolemia and studies of sterol balance.

We have evaluated the efficacy of plant sterol preparations from two different sources and in two different physical forms in lowering the plasma cholesterol of a total of 46 patients with type II hyperlipoproteinemia when given in addition to appropriate diet therapy. In addition, the mechanisms of the hypocholesterolemic effect were investigated in 7 patients by a sterol balance technique. The maximal mean cholesterol lowering in response to any preparation was 12 percent, although it was much greater in some individual patients. Sterol balance data showed that plant sterols inhibit cholesterol absorption with maximal negative cholesterol balance in adults at a dose of 3 g/day of a tall oil sterol suspension. Interestingly, maximal plasma cholesterol reduction in the adult outpatients on this preparation was seen at the same dose level. Since the tall oil sterol suspension is relatively palatable and is poorly absorbed, it has potential value as an adjunct to dietary therapy in patients with mild hypercholesterolemia for whom long-term drug therapy is deemed advisable.


Occurrence of individual trans-isomeric fatty acids in human myocardium, jejunum and aorta in relation to different degrees of atherosclerosis.

With the aid of a highly selective capillary column coated with SP 2340, individual trans-fatty acids were identified in all tissue samples studied, the values being unrelated to the degree of atherosclerosis. The mean values of total trans-acids present in human myocardium, jejunum and aorta amounted to less than 1% of the total fatty acids. The trans-acid content was composed of traces of 9-trans-tetradecenoic acid and nearly equal amounts of 9-trans-hexadecenoate and trans-octadecenoate, the double bond in the latter occurring in the delta6, delta9 and delta11 positions. 9-trans,12-trans-octadecadienoate or the corresponding mixed geometric isomers could not be identified in any sample. Additionally, significant increases of 20:4c(n-6), 22:4c(n-6) and 22:6c(n-3) acids were measured in the myocardium of patients who had died from severe atherosclerosis of the aorta and coronary arteries. The atherosclerosis was not attributable to different dietary habits, as judged from the content of linoleic acid in their adipose tissue.


Regression of atheroma in the rabbit.

Ten studies in the literature concerning regression of rabbit atheroma were re-examined. In studies where cholesterol content was referred to weight, a degree of regression was noted in 3/4 studies. Such regression might at least partly have resulted from the dilution effect of the atheroma contents when results were expressed on a weight basis. By contrast, when results were referred to length or protein, partial regression was seen in only 1/4 studies. Mild atheroma induced by short-term cholesterol feeding did seem to regress in 2/2 studies.


Serum lipids and coronary heart disease in heterozygous familial hypercholesterolemia in the Hokuriku District of Japan.

The serum cholesterol and triglyceride levels and the incidence of ischemic heart disease were studied in 122 (55 men and 67 women) consecutive heterozygous familial hypercholesterolemic patients in the Hokuriku district of Japan. (1) The mean +/-SD of serum cholesterol level was 354.0 +/- 71.0 mg/100 ml, which was lower than those of the Western countries by about 60--70 mg/100 ml. (2) The mean +/-SD of serum triglyceride level was 116.5 +/- 54.0 mg/100 ml. (3) The average serum cholesterol values in the 20--50-year-old group showed no differences from those of the Western countries. However, in the above 50 years of age group the serum cholesterol levels were much lower than those in the United States. (4) The occurrence of ischemic heart disease in 83 heterozygous familial hypercholesterolemic patients was 43.3%. The incidence of myocardial infarction was 20.5%. Thus, familial hypercholesterolemia is as highly atherogenic as that of the Western countries even in Japan where the low incidence of coronary heart disease in the general population has been attributed to the low level of serum cholesterol.


Removal of fatty acid labelled cholesterol ester, phospholipid and triglyceride from atherosclerotic rabbit aorta in vitro.

An organ culture technique was used to investigate the removal of fatty acids incorporated into various lipid fractions in intimal segments of atherosclerotic rabbit aorta. Segments of aorta were pulse-labelled in vitro with [9,10-3H]oleic acid and [1-14C]linoleic acid for 24 h. The incubation medium was replaced with non-radioactive medium and the removal of the endogenously labelled phospholipid, triglyceride and cholesterol ester was followed for a further period of 10 days. Fatty acids incorporated into phospholipid and triglyceride were removed rapidly from the aortic intima, with fractional half-times of 4--5 days. Cholesterol ester fatty acids were removed more slowly from the aortic intima with fractional half-times of 9--12 days. When the removal rate of oleic acid incorporated into phospholipid, triglyceride and cholesterol ester was compared with that of linoleic acid incorporated into the same lipid fractions, no difference between removal of the two fatty acids could be detected with respect to any of the 3 lipid fractions.


Strain differences in response to dietary cholesterol by JAX rabbits: correlation with esterase patterns.

Six strains of genetically defined JAX rabbits were tested for their serum cholesterol levels (total and free) in response to a 0.5% cholesterol diet. Marked differences in response between the 6 strains were found. IIIVO/J and WH/J are low responding strains, X/J and ACEP/J are intermediate responding strains, and OS/J and AX/J are high responding strains. After 4 weeks of the cholesterol diet the total serum cholesterol level of the high responding AX/J strain was about 5-fold greater than the level of the low responding IIIVO/J strain. The esterified/total (E/T) ratio appeared to be higher in the high responding strains, indicating a synergistic effect in the process of atherosclerosis. The response of the individual rabbits to the cholesterol diet was compared with the patterns of serum and liver esterase zymograms. This comparison indicated a correlation of the dietary cholesterol susceptibility with the presence or absence of the esterase zones in the anodal, fast moving region of the gel.


Studies of the mechanism of augmented synthesis of cholesteryl ester in atherosclerotic rabbit aortic microsomes.

A study was undertaken to test the hypothesis that an abnormally high concentration of acyl-CoA:cholesterol acyltransferase in atherosclerotic microsomes is partly responsible for augmented esterification of cholesterol. We approached the problem indirectly by measuring the incorporation of radioactivity into cholesteryl ester from [1-14C]palmityl-CoA in normal microsomes after enrichment of their concentration of microsomal free cholesterol to levels characteristic of atherosclerotic microsomes. Elevation of free cholesterol content induced increased cholesterol esterification approximately linearly over the range studied. The cholesterol-esterifying activity of atherosclerotic microsomes was not greater than that of normal microsomes having the same concentration of cholesterol. The results suggest that, with acyl-CoA constant, augmented cholesterol esterification in atherosclerotic microsomes is an effect of high microsomal cholesterol concentrations and not due to an increase in the concentration of the enzyme.


Induction of aortic lipid deposition in a high-response (ExHC) rat fed a diet containing cholesterol and cholic acid.

Male ExHC and Sprague--Dawley rats were fed a diet containing 3% cholesterol, 0.6% sodium cholate and 15% olive oil for 16 weeks. The ExHC rat is highly susceptible to dietary hypercholesterolemia. Aortas of the ExHC rats showed Sudan-stained deposits and cholesterol accumulation in the intima and media, whereas no deposited lipids were seen in those of the Sprague--Dawley rats. In male and female ExHC rats fed diets containing the above supplements at 3 dose levels, plasma cholesterol and aortic lipid deposition were found to be dose-dependent. Lipid deposition was more prominent in female than in males, but aortic intimal proliferation was absent.


A scanning electron microscopic study of arterial endothelial cells using vascular casts.

Vascular casts were made of rabbit aortas by infusing Batson's No. 17 anatomical corrosion compound into the artery at physiological pressure. The arterial tissue was then digested with sodium hydroxide and the cast viewed by scanning electron microscopy (SEM). Outlines of the endothelial cells and their silver stained boundaries were clearly visible. Cell nuclei and fine surface detail were also discernible. In EDTA damaged arteries, injured endothelial cells and platelets could also be observed in the vascular casts.


Calculation of the individual and population doses on Danish territory resulting from hypothetical core-melt accidents at the Barsebäck reactor.

Individual and population doses on Danish territory are calculated from hypothetical, severe core-melt accidents at the Swedish nuclear plant at Barsebäck. The release fractions for these accidents are taken from WASH-1400. Based on parametric studies, doses are calculated for very unfavourable, but not incredible weather conditions. The probability of such conditions in combination with wind direction towards Danish territory is estimated. Doses to bone marrow, lungs, GI-tract and thyroid are calculated using dose models developed at Risø. These doses are found to be consistent with doses calculated with the models used in WASH-1400.


Structural basis for aconitase activity inactivation by butanedione and binding of substrates and inhibitors.

Aconitase (citrate(isocitrate)hydro-lyase, EC 4.2.1.3) prior to activation demonstrates a single binding site for substrates and inhibitors. On the basis of kinetic experiments, at pH 8.5 and 37 degrees C, with monomeric butanedione in borate, this binding site was found to contain a single arginine residue. Dissociation constants at pH 8.5 and 37 degrees C, determined from inhibitory effects on butanedione inactivation rates are: citrate, 0.74 mM; D-isocitrate, 0.33 mM: cis-aconitate, 0.52 mM; tricarballytate, 0.42 mM; trans-aconitate, 0.025 mM. Corresponding dissociation constants for the active enzyme are: tricarballylate, 0.39 mM; trans-aconitate, 0.14 mM. Active site Fe2+ added to the enzyme on activation is therefore not required for binding. Km values are: citrate, 0.23 mM and cis-aconitate 0.012 mM. Binding to active enzyme is considered to be transition state binding.


Purification of commercial acriflavine by Sephadex LH-20 column chromatography.

Acriflavine supplies commercially was purified by removing contaminating proflavine by chromatography on a column of Sephadex LH-20 eluded with 65% methanol. The purified acriflavine, recovered in a yield that was 52% of the theoretical maximum, had a visible absorption maximum of 452 nm with a band width of 47 nm. These spectral properties agreed with those of acriflavine purified by precipitation, repeated recrystallization and chromatography on aluminum oxide, which results in a yield of 13% at best.


Use of high dose corticosteroids in patients with inoperable brain tumours.

Eleven patients with inoperable brain tumours were treated with high doses of corticosteroids (methylprednisolone 200-2000 mg/day) for up to 151 days (mean 55 days). Neurological improvement occurred in eight patients on high doses after deterioration on concentional doses (methylprednisolone 80-120 mg/day). In two patients steroids could be completely discontinued for several months. Serious adverse effects included sepsis in three patients and myopathy in tow. All three patients with sepsis also received chemotherapy. There were no deaths that could be attributed to steroids. The most likely effect of high dosage steroids is reduction of cerebral oedema. It is conceivable that in some instances high dose steroids may also result in tumour inhibition or oncolysis or both.


Increased incidence of renal cell carcinoma with hypertension.

Of 276 patients who underwent evaluation to determine the cause of hypertension 2 were found to have unsuspected renal cell carcinoma. This prevalence of 0.73 per cent is 16 times higher than expected for an age-matched population. Both patients exhibited abnormalities of plasma renin activity before radical nephrectomy but they remained hypertensive postoperatively. The data suggest an unexplained association between hypertension and renal cell carcinoma. Furthermore, abnormalities on excretory urograms in hypertensive people should be defined by selective renal arteriography.


Early detection of colonic neoplasia in patients at high risk.

An abnormal zone of DNA synthesis at the surface and upper portion of colonic crypts has been thought to be related to future adenomatous polyp development and to express a regulatory defect in the mechanism that normally terminates synthesis in the upper third. As part of a screening program for early colon cancer detection, patients over 40 years of age found to have occult blood in their stool (Ho+) are evaluated by barium enema and colonscopy as well as isotopic incorporation studies of biopsy and lavage specimens. This proliferative abnormality occurred most frequently among patients with an adenoma or adenocarcinoma although the frequency varied among simultaneous biopsies from the same patient. Specimens from Ho+ patients with a tumor often contained small areas of focal atypism in the biopsy or fragments of atypical epithelial cells in the lavage sample. A small group of Ho+ patients in whom no overt neoplasm could be detected also demonstrated surface-labeled epithelial cells with morphological alteration of these cells. Based on the microscopic findings presented, continued surveillance of these patients is suggested, as well as extension of these studies to include other high risk groups.


Pleural fluid complement, complement conversion, and immune complexes in immunologic and nonimmunologic diseases.

Forty-four pleural fluids and 41 blood specimens from patients with various diseases were examined for concentration of whole complement, C4, C3, conversion products of C3 and C3PA, and immune complexes. C3 conversion was found in all eight pleural fluids from patients with rheumatoid arthritis, five of seven with lupus erythematosus, two of six with congestive heart failure, and nine of 23 with malignant diseases. Conversion of C3PA correlated closely with C3 conversion and both were significantly inversely related to whole complement, C4, and C3. Concentration of immune complexes was highest in patients with rheumatoid arthritis. Pleural fluid immune complex concentrations correlated positively with conversion of C3 and C3PA. These findings suggest that the reduced levels of pleural fluid complement in rheumatoid arthritis and lupus erythematosus may be secondary to complement conversion by immune complexes.


DDVP (dichlorvos) detoxification by binding and interactions with DDT, dieldrin, and malaoxon.

Binding to tissue carboxylesterases has been suggested as an important mechanism of detoxification for several organophosphates. In this study DDVP, malaoxon, and paraoxon were inactivated, in vitro, by mouse liver under assay conditions that were consistent with a binding mechanism of inactivation. Binding of the three organophosphates was inhibited in livers of mice pretreated, 18 hr before sacrifice, with TOTP (triorthotolyl phosphate, 125 mg/kg, ip). Previous studies have shown that similar TOTP treatment enhanced the toxicity of malaoxon and paraoxon but did not alter the toxicity of DDVP. Both DDT (50 mg/kg, ip, given 4, 3.5, and 2.5 days before sacrifice) and dieldrin (16 mg/kg, po, given 4 days before sacrifice) increased liver/body weight ratios and decreased the duration of pentobarbital-induced loss of righting ability. Dieldrin increased liver carboxylesterase activity and liver binding of malaoxon and paraoxon but not of DDVP. In contrast, DDT did not increase liver carboxylesterase activity nor did it increase binding of malaoxon or paraoxon. Yet DDT pretreatment increased mouse liver binding of DDVP. Neither DDT nor dieldrin pretreatments altered the toxicity of subsequently administered DDVP. These results support the hypothesis that carboxylesterase binding does not represent an important mechanism for DDVP detoxification in the mouse. In other experiments, in vitro inactivation of malaoxon by binding was inhibited in livers of mice given DDVP (30 mg/kg, ip) 30 min before sacrifice. Similar DDVP pretreatment potentiated the anticholinesterase action of malaoxon (10 mg/kg, ip). Thus, even though DDVP toxicity was not altered by DDT- or dieldrin-induced changes in organophosphate binding, DDVP inhibited malaoxon binding and increased malaoxon toxicity.


Ventilatory effects accompanying spontaneous movement in the suckling opossum.

To determine whether spontaneous movement was associated with an organized respiratory response during early stages of mammalian development, we studied immature suckling opossums (Didelphis virginiana) between 30 and 60 days of age. Rate and depth of breathing were measured using pressure plethysmography and movement was assessed using electromyography of neck muscles. During air-breathing as well as during ventilatory chemostimulation with hypoxic, hypercapnic, and asphyxiant test gases, the onset of spontaneous movement was often marked by an increase in minute-ventilation, dominated by an increase in breathing frequency. In the absence of respiratory irregularities, ventilation and breathing frequency remained elevated throughout an episode of movement. After decerebration at the midcollicular level, the onset of spontaneous movement was accompanied by respiratory effects similar to those found in the intact preparation. The ventilation response with spontaneous movement contrasted to results during venilatory chemostimulation at rest, where increases in breathing resulted primarily from increases in tidal volume. It is concluded that a hyperpnea with spontaneous movement is present in the suckling opossum, and that the effect is primarily organized in the lower brainstem.


Heterakidosis and nodular granulomas caused by Heterakis isolonche in the ceca of gallinaceous birds.

Five pheasants and two ocellated turkeys from the San Diego Zoological Garden had multiple cecal nodules. These nodules were composed mostly of granulomas and fibrous hyperplastic tissue associated with Heterakis isolonche.


Breast cancer and religion in greater Bombay women: an epidemiological study of 2130 women over a 9-year period.

The resident female population of Greater Bombay consists of women professing different religious faiths, between which the frequency of breast cancer varies to a great extent. During the 9-year period 1964 to 1972 inclusive, a total of 2130 women with breast cancer were seen, with break-down by religion as follows: Hindus (1259), Muslims (306), Christians (264), Parsi (Zoroastrians) (226), Jains (25), Buddhists (26) and others (24). The average annual age-adjusted (world population) incidence rates, however, were found to be 48.5 and 18.2 per 100,000 in the Parsis and non-Parsis respectively, with an average of 19.9 per 100,000 for the total population. For reasons not yet clear, in every age group the incidence rate in Parsis was 2 to 3 times higher than in the non-Parsis. Time-trend analyses of our data do not reveal any statistically significant increase or decrease in the incidence of breast cancer in any particular age group. Data from death certificates for the same 9-year period show that the age-adjusted mortality rate (world population) is 9.2 per 100,000/year.


Acute gastric dilatation associated with gastric volvulus in the guinea pig.

During an 18-month period, gastric dilatation was recognized in six guinea pigs from a colony with a monthly average of 253 animals. All of the affected guinea pigs were breeders and ranged in age from 8 to 26 months. Clinical signs were tachycardia, dyspnea, cyanosis, and marked abdominal distention. Macroscopic lesions included gastric volvulus, gastric dilatation, and splenic congestion.


Basal cell nevus syndrome.

We report two patients with basal cell nevus syndrome. They had two previously unreported associated conditions--a myocardial fibroma and bilateral basal cell carcinomas of the soles of the feet. The syndrome is discussed, the literature is reviewed, and the management is outlined.


[Studies of influenza in Palermo, 1970-75].

Epidemiological surveillance of influenza was carried out in Palermo betwen 1970 and 1975. Antigenic structure of viral strains A/H3N2 isolated from influenza patients during winter outbreaks was studied in comparison with reference strains circulating in the same periods in other parts of the world. Antigenic drift of hemagglutinin, of neuraminidase, or of both, was observed in Palermo isolates, which were progressively different from Hong Kong/68 strain; 1973 and 1975 isolates were similar to A/England/72 and A/Port Chalmers/73. HI antibodies against Palermo isolates were titered in serum samples obtained from adults living in Palermo. Seronegativity for each strain progressively decreased from 1971 to 1975.


[Optimal temperature of enzyme activity and heat resistance of neuraminidases as attenuation markers for influenza viruses].

Neuraminidase activity of virulent and vaccinal strains A/England/42/72, A/Scotland/840/74 and A/Victoria/3/75 assessed at different temperatures did not show significant differences in the temperature optima of enzyme activity. Vaccinal strains were attenuated by APR-8 recombination and selection of mutants resistant to serum inhibitors (Alice, RIT 4025, RIT 4050 strains). On the contrary, differences were found in the thermal inactivation of the neuraminidase between wild and vaccinal strains, the latter being more thermolabile. Increasing from 0.5 to 5.0 mM/1 the Ca++ concentration, an enhancement of the heat stability was observed, more marked for the attenuated strains. The amount of NANA released by 1 hemagglutinating unit varied widely between strains. To be pointed out, however, that within each antigenic variant the vaccinal strain showed a lower enzymatic activity than the corresponding wild strain.


[Studies of an epidemic episode of serous meningitis caused by ECHO-virus 30].

The Authors report an outbreak of ECHO 30 aseptic meningitis occurred in Marche and Umbria countries from November 1975 to June 1976. The clinic, epidemiologic characteristics and the serological and virological results of 28 cases are discussed.


Leptospiras as causative organisms of meningitis.

Data concerning the incidence of meningeal syndromes during the course of leptospirosis are analyzed, in relation to the Italian epidemiological situation in the years 1973-1976. From this analysis, some characteristic aspects concerning meningitis from leptospiras emerge. Specifically: the remarkable frequency of meningeal syndromes in leptospirosis; the greater incidence of the syndrome among young people; the neurotropism common to all serotypes of leptospira observed in Italy. These data suggest to extend the search for leptospiroses in all forms of the so called aseptic meningitis in order to determine better the actual incidence of leptospiroses in meningeal syndromes.


Environmental quality induced predicted by evolutionary theory.

The use of evolutionary theory to understand and predict environmental quality indices has been described. Three specific examples concerning the environmental health implications of black skin, heat stress adaptation, and G-6-PD deficiency have been discussed in which evolutionary theory provides the conceptual framework for novel insights and reasonable predictions of environment, pollutant and human interactions. The use of the evolutionary paradigm in the development of environmental quality indices offers a clear alternative to trial and error research efforts.


[Changes in the vaginal mucosal bacterial flora ecosystem during the menstrual cycle in women].

It was shown that bacteria of mucosae of higher organisms, mostly have specific receptors for chemical complementary structures of cell surface. On the other hand it is known already that vaginal flora changes during ovulation and afterwards, some infections begin and reveal during some periods of the ovulatory cycle. Therefore, it was studied the adherence of saprophytic and pathogenic bacteria to vaginal cell in different phases of the ovulatory cycle. It was shown that, during the cycle, the bacterial adherence changes because of environment modifications, caused by secretion under hormonal control.


Enhanced early response to tetanus toxoid, in rabbits.

AIPO4 adsorbed tetanus toxoid injected in rabbits according different vaccination schedules, gives rise to antibody responses with varying lag periods. Shortening the intervals among antigen injections, or increasing the quantity of toxoid per dose, makes it possible to reach protective serum antibody levels in 10 days. That is to say, in a period of time shorter than the mean tetanus incubation in man. Although difficult to transfer to man, the exposed data suggest a possible differentiated antitetanus vaccination in subjects at risk, non protected.


IgG and C3 receptors on polymorphonuclear leukocytes in phagocytosis. Role of C3-receptors in the attachment and ingestion phases.

The role of IgG and complement (C3) receptors in the adhesion and ingestion phases of immune complexes by normal human polymorphonuclear leukocytes (PMN) were examined. The immune complexes were sheep red cells (E) sensitized with IgG (EA) or IgM antibodies plus complement (EAC'). Both types of receptor sites are involved in the adhesion phase. Moreover IgG receptors are primarily involved in the ingestion phase. Nevertheless even C3-receptors may be sufficient for complete phagocytosis. Even if EAC' adhesion was still high, C3-receptor specific phagocytosis decreased parallel with the amount of the complement used for EA19S sensibilization. The role of receptor sites on human PMN in adhesion and ingestion phases is discussed.


[Evaluation of the cell-mediated immune response after administration of Salmonella antigens in patients with multiple myeloma].

Myeloma patients show an impairment of several immunological functions leading to infectious diseases. In this study we evaluated cell-mediated responsiveness in a group of these patients. Six myeloma patients received full doses of I.S.M. Enterovaccino. LIF production by peripheral blood lymphocytes against Salmonella antigens was evaluated after 14 days. All patients studied showed no LIF production.


[A case of cerebral arteriovenous malformation (AVM) with presented remarkable enlargement during 13 years (author's transl)].

A 20-year-old man visited us complaining of head heaviness after he sustained a minor head injury at traffic accident. Although he showed no neurological deficits, the CT--SCAN (Fig. 2) revealed a high density area after contrast enhancement in the left frontal region. Left carotid angiography (Fig. 3) was carried out on admission, revealing a large AVM (3X5X4 cm) in the same area. As he had undergone craniotomy because of chronic subdural hematoma at the age of seven, the left carotid angiogram (Fig. 1) taken at that time was re-examined. It revealed no abnormal shadows suggesting the presence of the AVM. Since AVM is a congenital disease, the present large AVM is considered to have grown in size from the cryptic type for the past 13 years, escaping detection by the initial angiogram.


[Radiological anatomy of the anterior cerebral vein (author's transl)].

The anatomy of the anterior cerebral vein is described with injection specimens. Radiological appearance of the anterior cerebral vein is illustrated with conventional angiograms. Stereoscopic films are indispensable to identify the anterior cerebral vein accurately. The anterior communicating vein or the distal end of the anterior cerebral vein indicates the anterior wall of the lower portion of the third ventricle. The anterior cerebral vein is very useful in defining the suprasellar extension of an intrasellar mass.


Inhibition of phagocytosis and glucose metabolism of alveolar macrophages during pulmonary tumour growth.

Alveolar macrophage (AM) phagocytic activity and glucose metabolism were evaluated during lung tumour growth in adult rats challenged i.v. with 10(5) viable Walker 256 tumour cells. Phagocytosis was estimated by the in vitro uptake of (14)C-labelled Pseudomonas aeruginosa and glucose oxidation was evaluated by (14)CO(2) production from 1-(14)C-glucose. AM were harvested by lung lavage from rats prior to and at 7 and 21 days following i.v. tumour-cell challenge. Macroscopic lung tumour nodules were not observed by 7 days after tumour challenge. However, 3 weeks after tumour challenge, tumour nodules were clearly identifiable on the surfaces of the lungs. One week after the i.v. tumour challenge a marked increase in the number of AM was evident. The in vitro phagocytosis of (14)C-labelled Pseudomonas aeruginosa was unaltered at that time, but became progressively depressed thereafter. Three weeks after tumour challenge, this decrease in phagocytic activity was evident when cells were incubated in normal serum, and was furtheri ntensified by serum obtained from tumour-bearing animals. Glucose oxidation by AM in either the resting condition or during bacterial phagocytosis was clearly decreased at both 1 and 3 weeks following i.v. tumour challenge. These findings indicate that the growth of pulmonary metastases is associated with a depression of alveolar macrophage bacterial phagocytic capacity, perturbations in serum opsonic activity and distinct alterations in macrophage energy metabolism. The metabolic dysfunction may impair pulmonary macrophage host defences against lung tumour growth.


Detection of sensitized human blood lymphocytes by agglutination with basic peptides: a possible test for malignant disease.

A simple rapid micro-agglutination test for detecting sensitized lymphocytes from human peripheral blood is described: the poly-L-lysine (PLL) 3400 agglutination test of lymphocytes (PAL test). The incubation of lymphocytes from 30 cancer patients, 30 patients with non-malignant disease and 40 healthy controls with PLL (mol. wt 3400) is evaluated. The test was positive in 83 percent of malignant and 20 percent of non-malignant cases. All healthy controls were negative. Other peptides tested showed no significant difference in reaction between malignant and non-malignant diseases. The mechanism of detection of sensitized lymphocytes from patients with malignant disease through agglutination induced by PLL 3400 is discussed.


In vitro immune responses to PPD, extracts from Raji cells and nasopharyngeal carcinoma biopsies in NPC leucocytes.

Peripheral leucocytes from nasopharyngeal carcinoma (NPC) patients and control subjects, which included healthy subjects and patients with other cancers, have been tested against PPD and a panel of extracts from Raji cells and pooled NPC biopsies, using the blast transformation and the macrophage migration inhibition assays. The results of both assays indicated that the in vitro cell-mediated immune (CMI) responses to the Raji-cell extracts and NPC-biopsy extracts were associated with NPC. However, the peripheral leucocytes from NPC patients and control subjects responded similarly to PPD. These results are in general accord with the skin tests reported by Levine et al. (1976) and Ho, Ng and Kwan (1977b). The antigenic specificity of the NPC-associated CMI responses remains, however, to be resolved, as the extracts used in these or in the in vivo CMI studies were heterogeneous mixtures.


Lymphocyte function and response to chemo-immunotherapy in patients with metastatic melanoma.

Thirty-eight patients with metastatic melanoma were investigated for lymphocyte function immediately prior to chemo-immunotherapy. The pre-treatment immune tests were compared with normal control values and with response to therapy. The "non-responder" group (but not "responder") had significantly reduced values for lymphocyte, null-cell and E-rosette-cell counts compared with controls. Lymphocytoxicity ( using a Chang target cell) showed the same pattern, with depression of direct and K-cell cytotoxic capacity in non-responders compared with controls. Eight patients were studied sequentially whilst on treatment, and demonstrated considerable change (not statistically significant) in lymphocytotoxicity, an untreated "control" patient showed little variation. "Recall"-antigen skin testing showed no statistically significant difference between the patient groups. The data indicate that "non-T-cell activity" may be associated with response to chemo-immunotherapy.


Mononuclear cell-membrane "fluidity": a study in some haematological malignancies.

The polarization of fluorescence from diphenyl hexatriene embedded in the membranes of intact peripheral-blood mononuclear cells has been measured and used to assess the "microviscosity" or fluidity of the membrane. Cell preparations were examined from patients with various types of leukaemia and related conditions in which circulating primitive cells may occur. Significantly lower fluorescence polarization values were obtained in all samples from patients with chronic lymphocytic leukaemia, but normal results were obtained in cases of chronic granulocytic leukaemia, myelosclerosis, solid lymphomas and in acute leukaemias in remission. In relapsed acute leukaemia, fluorescence polarization indicated reduced "microviscosity" of the cell membrane when large numbers (greater than 10(9)/1) of primitive cells were present; normal "microviscosity" was indicated when less than 10(9)/1 primitive cells were present. However, exceptions occurred in both cases, and the technique failed to give warning of imminent relapse in one case. Our findings suggest that a reduction in "microviscosity" as indicated by this technique is not a general property of the blood leucocytes in all types of leukaemia, and that the technique cannot, at present, be regarded as an alternative method for detecting circulating primitive cells.


Effect of Corynebacterium parvum on peripheral blood platelets.

The level of peripheral blood platelets was determined after i.v. injection of Corynebacterium parvum in normal C57BL mice and in those bearing the Lewis lung carcinoma. Twenty minutes after injection of a formalin-killed active strain (CN6134, (CN6134, which inhibited tumour metastases) or a killed inactive strain (CN 5888, which did not inhibit metastases) the number of circulating blood platelets was reduced by 50%. The level of platelets returned to control values by 8 h after the active, and by approximately 3 days after the inactive strain. The active strain alone caused a second and prolonged fall in platelet numbers, from approximately 16 h to 21 days after injection. Heparin given 3 X weekly to these mice restored the platelet count to normal values by 10 days after injection of active-strain C. parvum. The level of platelets in tumour-bearing mice was essentially similar to that in normal mice. Possible causes of the thrombocytopenia and the significance of platelets in metastasis are discussed.


Nasopharyngeal cancer in a total population: selected clinical and epidemiological aspects.

A nationwide study of nasopharyngeal cancer in Israel, during a 9-year period (1960-68) demonstrated a mean annual incidence rate of 1.0 per 100,000 in males and 0.4 per 100,000 in females. Significantly higher incidence was observed in residents born in North Africa (3.0 in males and 1. 1 in females). Survival was relatively better in females among patients with lymphoepithelioma and in those without neurological complications. The results may support the presence of environmental factors in aetiology, though genetic predisposition cannot be ruled out.


Influence of height, weight and obesity on risk of breast cancer in an unselected Swedish population.

A number of recent studies have shown an association between breast-cancer risk and height, weight and dietary habits, especially fat consumption. In the present study, height and weight were determined for 179  consecutive, unselected, breast-cancer patients and age-matched controls selected from a computerized population register. Height and weight for these two groups were compared, including two different indices for overweight (Quetelet's index and Broca's index). Comparisons were repeated after subdivision into pre- and postmenopausal women. In all calculations, the mean values of patients and controls were very similar and without significant difference. It therefore seems improbable that increased height and weight or obesity constitute risk factors for breast cancer. Earlier studies may have shown differences as the result of selection mechanisms not present in this study.


Malignant melanoma and oral contraceptive use among women in California.

Women who had used oral contraceptives, particularly long-term users, were found to have higher rates of malignant melanoma and of a past history of skin cancer than those who had never used oral contraceptives. This excess was confined to lesions of the lower limb. The association between oral contraceptive use and melanoma was noted in 3 separate sets of data, although it was statistically significant only in one. The possibility that this relationship is indirect because, for example, oral contraceptive users are more likely than never-users to be exposed to sunlight and thus to develop malignant melanoma, cannot be excluded.


Altruism and the practice of medicine.

Biological and psychological research into the antecedents of altruism has considerable significance for those involved in the teaching or practice of medicine. The evidence available indicates that altruistic behaviour is a universal phenomenon influenced by intra-individual, interpersonal, situational and sociocultural factors. A central theme which emerges is the strikingly ambivalent nature of the altruistic drive. The factors which may facilitate or inhibit altruism in medical students and doctors are discussed. Some ways of ensuring that medical training does not seriously distort altruism are suggested.


Psychotherapy: theory, experience, and personalized actuarial tables.

This paper addresses the issue of the role of theory in the actual application of psychotherapeutic operations. Within the present framework, psychotherapeutic effectiveness is seen as an empirical, actuarial process which occurs in an interpersonal setting separate from theoretical considerations. The role of theory is discussed and a rationale for the coexistence of equally 'effective' contradictory theories is presented. Suggestions for future research in the area of behaviour change are made and an argument for the eventual development of a 'therapeutic cookbook' is presented.


Voice analysis for the measurement of anxiety.

A recently developed technique for the acoustical analysis of speech is described. Speech is analysed electronically for the presence or absence of a microtremor having a frequency of about 10 Hz. This tremor is said to be attenuated in states of psychological stress. The paper present data supporting the validity of this as a measure of anxiety, using states of both normal and pathological anxiety. An objective scoring system is proposed to overcome some of the problems of unreliability. A number of practical advantages of the voice technique are described.