The interaction of alpha-N-(p-toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester with thrombin and trypsin. The syntheses are described of p-guanidino-L-phenylalanine and some of its derivatives. alpha-N-(p-Toluenesulphonyl)-p-guanidino-L-phenylalanine methyl ester is an excellent substrate of bovine trypsin (EC 3.4.21.4) (Km 57 micron; kcat. 320s-1 at pH 7.4-8.0) and a very poor substrate of human thrombin (EC 3.4.21.5) (Km 190 micron, kcat. 0.2s-1) and bovine chymotrypsin (EC 3.4.21.1). The ester inhibits thrombin clotting activity. It also inhibits the amidase and esterase activities of human thrombin, this inhibition being of the mixed type. The inhibition constant, K1, of the order of 1 micron, increases with increasing inhibitor concentration. This suggests that the enzyme binds the inhibitor at multiple sites. The importance of the residue at the P1 position [notation of Berger & Schechter (1970) Philos. Trans. R. Soc. London Ser. B 257, 249-264] in determining the selectivity of a substrate or quasi-substrate among trypsin-like enzymes is borne out. p-Guanidino-L-phenylalanine may have a use in the synthesis of selective peptide inhibitors of thrombin.


Actin in young and senescent fibroblasts. Double labelling and the isolation of peptides specific to muscle actin were used to determine the amount of actin in young and senescent fibroblasts from chicken embryo. A 20-residue peptide produced from the C-terminus of muscle actin was found in amounts that indicated 6.9% of the protein of young fibroblasts and 11.5% of the protein of senescent fibroblasts to be actin. Two other peptides of muscle actin were present in very small amounts. This indicates that the actin of both young and senescent fibroblasts is homologous to, but not identical with, muscle actin. Increased actin content of senescent fibroblasts may be related to their loss of proliferative capacity and low cell density at confluence.


The binding of 2,4,6-trinitrophenyl derivatives to the mouse myeloma immunoglobulin A protein MOPC 315. The binding of Tnp (2,4,6-trinitrophenyl) derivatives to the Fv fragment (variable region of heavy and light chains) of the mouse myeloma IgA protein MOPC 315 was investigated by 270MHz proton nuclear magnetic resonance. Two of the haptens, Tnp-glycine and Tnp-l-aspartate, are in fast exchange with the Fv fragment, and the changes in chemical shifts for both protein and hapten resonances were determined by titrations. For the tightly binding hapten epsilon-N-Tnp-alpha-N-acetyl-l-lysine, which is in slow exchange with the Fv fragment, the changes in chemical shifts for the hapten H(3)+H(5) resonances were determined by cross-saturation. By using these data and the known structure of the combining site of protein MOPC 315 [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977), Nature (London) 266, 31-37] the mode of binding of Tnp derivatives is deduced by ring-current calculations. The trinitrophenyl ring stacks with tryptophan-93(L) (light chain) in the ;aromatic box' formed by tryptophan-93(L), tyrosine-34(L) and phenyl-alanine-34(H) (heavy chain). Further evidence for the stacking interaction with a tryptophan residue is provided by the similarity of the optical-difference spectra observed with Tnp-aminomethylphosphonate in the presence of either the Fab fragment (light chain and N-terminal half of heavy chain) of protein MOPC 315 or tryptophan. These data show that the modes of binding of all the Tnp derivatives are very similar, despite a 100-fold range in their affinities. It is also concluded that the modes of binding of Dnp (2,4-dinitrophenyl) and Tnp derivatives to protein MOPC 315 are very similar, and that the structural basis for this is that the aromatic box is large enought to allow the trinitrophenyl ring to stack with tryptophan-93(L) while still forming hydrogen bonds to asparagine-36(L) and tyrosine-34(L).


Further characterization of collagen galactosyltransferase from chick embryos. Optimum extraction of collagen galactosyltransferase activity from chick embryos required relatively high concentrations of detergent and salt. The activity was inhibited by concanavalin A, and the enzyme had a high affinity for columns of this lectin coupled to agarose; these results suggest the presence of carbohydrate units in the enzyme molecule. Collagen galactosyltransferase was highly labile, and only 1% of the originally bound enzyme activity could be eluted from the concanavalin A-agarose column with a buffer containing methyl glucoside and ethylene glycol. The purification of the activity over the original supernatant of chick embryo homogenate was 250-300-fold, with the optimum reaction conditions for the purified transferase differing somewhat from those for crude enzyme preparations. The reaction was inhibited by glucose-free basement-membrane collagen, UDP and galactosylhydroxylsine, and also by Co2+ and a number of compounds resembling UDP-galactose. Hydroxylysine was also a weak inhibitor. Immobilized hydroxylysine and UDP-glucuronic acid did not bind the collagen galactosyltransferase, but the enzyme was retarded in a column of UDP-galacturonic acid linked to agarose.


A radiochemical method for the measurement of coproporphyrinogen oxidase and the utilization of substrates other than coproporphyrinogen III by the enzyme from rat liver. [14C2]Coproporphyrin III, 14C-labelled in the carboxyl carbon atoms of the 2- and 4-propionate substituents, was prepared by stepwise modification of the vinyl groups of protoporphyrin IX. The corresponding porphyrinogen was used as substrate in a specific sensitive assay for coproporphyrinogen oxidase (EC 1.3.3.3) in which the rate of production of 14CO2 is measured. With this method, the Km of the enzyme from rat liver for coproporphyrinogen III is 1.2 micron. Coproporphyrin III is a competitive inhibitor of the enzyme (Ki 7.6 micron). Apparent Km values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9 micron and that for harderoporphyrinogen 1.6 micron. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroisocoproporphyrinogen at a rate similar to that for the formation of protoporphyrinogen IX from coproporphyrinogen III. Mixed-substrate experiments indicate that this reaction is catalysed by coproporphyrinogen oxidase and that the Km for this substrate is 29 micron. It is suggested that the ratio of the concentration of pentacarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX.


Factors determining the sequence of oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III by coproporphyrinogen oxidase in rat liver. Coproporphyrinogen oxidase (EC 1.3.3.3) catalyses the oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III to form protoporphyrinogen IX. A 4-propionate-substituted porphyrinogen, harderoporphyrinogen, which is also a substrate for coproporphyrinogen oxidase, is formed during the reaction. Synthetic [(14)C]coproporphyrinogens III, specifically labelled in the carboxyl carbon atoms of either the 2- or 4-propionate substituents, were used to measure the rate of decarboxylation of each substituent by rat liver coproporphyrinogen oxidase. The experimental results, together with the recognition that in all known substrates of coproporphyrinogen oxidase only those propionate groups flanked by a specific arrangement of substituents are decarboxylated, indicate that the 4-propionate group of coproporphyrinogen III cannot be attacked until the 2-propionate group has been decarboxylated. Production of (14)CO(2) from the substrate labelled in the 2-propionate group therefore measures the formation of harderoporphyrinogen, whereas (14)CO(2) from the 4-propionate-labelled substrate measures protoporphyrinogen IX formation. The rate of harderoporphyrinogen formation is about twice that of protoporphyrinogen, and this ratio is unchanged by varying the concentration of coproporphyrinogen III or by competitive inhibition of the enzyme. When coproporphyrinogen III is present in an excess, two fractions of harderoporphyrinogen can be distinguished. One accumulates during the reaction, and the other, which is destined to become protoporphyrinogen IX, does not equilibrate with added harderoporphyrinogen. It is suggested that both decarboxylations take place at the same active centre, which becomes temporarily inaccessible to coproporphyrinogen III and added harderoporphyrinogen, and that the molecule rotates after the first decarboxylation to allow the second to take place.


A reappraisal of the reaction pathway of pyruvate carboxylase. The reaction pathway catalysed by pyruvate carboxylase was re-examined by using two independent experimental approaches not previously applied to this enzyme. To avoid the variable stoicheiometry associated with oxaloacetate formation, the reaction rate was measured by following release of Pi. Initial velocities, when plotted as a function of varying concentrations of either MgATP2- or HCO3-, at fixed concentrations of pyruvate, gave in double-reciprocal-form families of straight intersecting lines. Further, when the reaction velocity was determined as a function of varying MgATP2- concentrations by using pyruvate, 3-fluoropyruvate and 2-oxobutyrate as alternative carboxyl-acceptor substrates, the slopes of the double-reciprocal plots were significantly different. Both results support a sequential reaction pathway.


The components of troponin from chicken fast skeletal muscle. A comparison of troponin T and troponin I from breast and leg muscle. The three components of troponin were prepared from chicken breast and leg muscle. The troponin I and T components were separated by chromatography on DEAE-cellulose after citraconylation and without the use of urea-containing buffers. The troponin I and C components were similar to their counterparts from rabbit fast skeletal muscle, and a comparison of the troponin I components from breast and leg  muscle by amino acid analysis, gel electrophoresis and peptide 'mapping' provides strong evidence for the identity of these proteins. The molecular weights of the troponin T components from breast and leg muscle were 33 500 and 30 500 respectively, determined by gel filtration. A comparison of these two proteins by methods similar to those used for the troponin I components suggested that they differed only in the N-terminal region of the sequence, the breast-muscle troponin T having an extra length of polypeptide chain of approx. 24 residues that is rich in histidine and alanine. The N-terminal hexapeptide sequence, however, is the same in both proteins and is (Ser,Asx,Glx)Thr-Glu-Glu. The genetic implications of these findings are considered.


Anomalous migration of leghaemoglobin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The estimate of the molecular weight of leghaemoglobin by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is about 20% too low. This is due to an anomalously high limiting relative mobility. Leghaemoglobin binds 1.4 g of sodium dodecyl sulphate/g of protein with a concomitant decrease in the helical content from 71-72% to 49-51%.


Use of flux ratio measurements for the determination of the order of addition of substrates and products in enzyme reactions. 1. Methods of determining the order of addition of substrates and dissociation of products by using flux ratios are investigated. Where an enzyme obeys hyperbolic steady-state velocity kinetics it is concluded that it may be particularly useful to compare the measured flux ratios with those calculated from the steady-state velocity parameters. 2. An expression is derived relating the relative contribution of the two pathways in a branched pathway to the flux ratios. 3. The relationship of equilibrium-reaction-rate measurements [Boyer & Silverstein (1963) Acta Chem. Scand. 17, Suppl. 1, S195] to the flux ratios is considered. Equilibrium-reaction rates are shown to be affected both by the addition of substrates and dissociation of products. Methods of analysing the data to distinguish between these events are discussed. 4. Methods of measurement of flux ratios are described, and it is concluded that the non-equilibrium steady-state method is preferable to measurements at chemical equilibrium. 5. The relative significance of flux ratio measurements and steady-state velocity inhibition data is discussed. It is concluded that flux ratios, when taken in conjunction with the inhibition data, provide the least ambiguous information about mechanism.


Kinetics and mechanism of action of muscle pyruvate kinase. 1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg(2+). 2. Methods of preparing [(32)P]phosphoenolpyruvate from [(32)P]P(i) in high yield and determining [(32)P]-phosphoenolpyruvate and [8-(14)C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [(32)P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-(14)C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [(32)P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg(2+), with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-(14)C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-(14)C]-pyruvate or [gamma-(32)P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.


Identification of the sites in collagen alpha-chains that bind serum anti-gelatin factor (cold-insoluble globulin). Anti-gelatin factor was prepared from guinea-pig and human serum by affinity chromatography on denatured type-I collagen. As shown previously, this component is related to cold-insoluble globulin. It reacted with 125I-labelled denatured collagen, and the reaction could be inhibited by preincubation with unlabelled collagenous components. In the inhibition assay comparable activities were observed for native and denatured type-I, -II, -III and -IV collagens. There was also no difference in reactivity between collagens of different species. The reactive sites in the collagen alpha-chains were located by inhibition assays on distinct CNBr- and collagenase-derived peptides. The results obtained with fragments from alpha1(I)-, alpha2- and alpha1(II)-chains indicate that the most active region is located between positions 643 and 819 of the alpha1-chain. Lower activities were found for other regions of collagen and may indicate that the factor has the potential to interact with several sites in the alpha-chains. The present data agree with observations by Kleinman, McGoodwin & Klebe [Biochem. Biophys. Res. Commun. (1976) 72, 426-432] on the specificity of a serum factor promoting the attachment of fibroblasts to collagen.


Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid. The biosynthesis of a hypermodified nucleotide, similar to or identical with 3-(3-amino-3-carboxypropyl)-1-methylpseudouridine monophosphate, present in Saccharomyces carlsbergensis 17S and HeLa-cell 18S rRNA, was investigated with respect to the sequence of reactions required for synthesis and their timing in ribosome maturation. In both yeast and HeLa cells methylation precedes attachment of the 3-amino-3-carboxypropyl group. In yeast the methylated precursor nucleotide was tentatively characterized as 1-methylpseudouridine. This precursor nucleotide was demonstrated in both 37S and most of the cytoplasmic 18S pre-rRNA (rRNA precursor) molecules. The synthesis of the hypermodified nucleotide is completed just before the final cleavage of 18S pre-rRNA to give 17S rRNA, so that the final addition of the 3-amino-3-carboxypropyl group is a cytoplasmic event. Comparable experiments with HeLa cells indicated that formation of 1-methylpseudouridine occurs at the level of 45S RNA and addition of the 3-amino-3-carboxypropyl group occurs in the cytoplasm on newly synthesized 18S RNA.


Localization of the ribonucleotide sites in rat liver mitochondrial deoxyribonucleic acid. Supercoiled rat liver mitochondrial DNA is relaxed by treatment with ribonucleases A, T1 or H. All the supercoiled mitochondrial DNA is sensitive to ribonuclease H and ribonuclease A, but only 35% of the supercoiled population is sensitive to ribonuclease T1. Removal of the ribonucleotides with calf thymus ribonuclease H, followed by denaturation of the mitochondrial DNA and analysis of the single-strand fragment lengths in the electron microscope, showed that the ribonucleotides were randomly located on both strands of the DNA. Endonuclease-S1 digestion of mitochondrial DNA after removal of the ribonucleotides reveals that no unique fragments are produced and ribonucleotides are randomly distributed with respect to one another. The average number of ribonucleotide sites per molecule was estimated to be between 8 and 13. Two possible mechanisms for the origin of ribonucleotide sites are discussed.


NAD(P)H dehydrogenase and its role in the vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone)-dependent carboxylation reaction. A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).


The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102. 1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.


Isolation, composition and ordering of cyanogen bromide peptides in human casein. The major component of the casein fraction of human milk was cleaved by cyanogen bromide, and the composition of the resulting peptides was determined. Casein was also subjected to limited digestion by trypsin, and the amino acid composition of the isolated peptides was established. With this information the peptides were ordered as they occur in the purified protein.


Purification and properties of a nuclear protein kinase associated with ribonucleic acid polymerase I. A cyclic AMP-dependent nuclear protein kinase was found to be closely associated with rat liver nucleolar RNA polymerase I throughout most of its purification. This protein kinase was purified to near homogeneity. It exhibits a number of unusual catalytic properties, including the inability to utilize Mn2+ when RNA polymerase is the substrate and the ability to phosphorylate both acidic and basic substrates. Phosphorylation of RNA polymerase I by this protein kinase results in the formation of phosphoester bonds characteristic of phosphoserine and phosphothreonine. Radioautography of polyacrylamide-gel electrophoretograms of the phosphorylated RNA polymerase I revealed that the 32P was located primarily on enzyme subunits SA1, SA3, SA5, and SA6 [nomenclature of Kedinger, Gissinger & Chambon (1974) Eur. J. Biochem, 44, 421-436].


Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking. 1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium  dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group.


Substrate specificity and mode of action of a cellulase from Aspergillus niger. The mode of action and substrate specificity of a cellulase purified from Aspergillus niger were examined. The enzyme showed little capacity to hydrolyse highly ordered cellulose, but readily attacked soluble cellulose derivatives and amorphous alkali-swollen cellulose. Activity towards barley glucan and lichenin was greater than with CM-cellulose. Low activity was detected with CM-pachyman (a substituted beta-1,3-glucose polymer) and xylan. Activity towards yeast glucan, mannan, ethlene glycol chitin, glycol chitosan, laminarin, polygalacturonic acid and pectin could not be demonstrated. Cellobiose and p-nitrophenyl beta-D-glucoside were not hydrolysed, whereas the rate of hydrolysis of the higher members of the reduced cellulodextrins increased with chain length. The central bonds of cellotetraosylsorbitol and cellopentaosylsorbitol were the preferred points of clevage. Kinetic data indicated that the specificity region of the cellulase is five glucose units in length. The evidence indicates that the cellulase is an endoglucanase.


An equilibrium study of metal ion binding to human plasma coagulation factor XIII. 1. The binding of Ca2+ to plasma coagulation Factor XIII from man and from cow caused a small decrease in the intrinsic fluorescence of the protein with a dissociation constant of 0.1 mM. A similar decrease was observed with the thrombin-activated Factors (Factors XIIa). The decrease in protein fluorescence was also caused by both Ni2+ and Mn2+ but not by Mg2+. 2. 45Ca2+ binding was directly demonstrated by equilibrium dialysis. Ca2+ at 0.2 mM bound to Factor XIII (a2b2) and Factor XIIIa (a'2b2) but not to isolated b2-protein. A tight-binding site for Ca2+ is associated with the a-subunits. 3. The Ca2+ essential for the enzyme activity of Factor XIII from man, pig and cow can be replaced by Ni2+, Cu2+, La3+, Mn2+, Fe3+, Y3+, Co2+, Sr2+ or Tb3+, but not by Mg2+.


Protein-protein interactions in blood clotting. The use of polarization of fluorescence to measure the dissociation of plasma factor XIIIa. 1. Human plasma Factor XIII (the precursor of fibrin-glutamine-fibrin-lysine endo-gamma-glutamyltransferase) was randomly labelled by incubation with fluorescein isothiocyanate. The biochemical properties of the system were unaltered by the label. The polarization of the fluorescein fluorescence attached to the plasma protein was measured and the following conclusions were reached. 2. Factor XIII (a'2b2) does not dissociate in the protein-concentration range 10-500 microgram/ml either with or without added Ca2+. 3. Factor XIIIa (a'2b2) does not dissociate in the absence of Ca2+ in the protein-concentration range 10-500 microgram/ml. 4. Additions of Ca2+ to Factor XIIIa result in a decreased polarization of fluorescence as the tetramer dissociates. The decrease in polarization was the same amplitude at protein concentrations 10-500 microgram/ml and Ca2+ concentrations 2-66 mM and indicates that the overall process is essentially irreversible. The decrease in polarization consisted of fast and slow exponential phases. Both the rate of the fast phase and the proportion of the reaction it represented increased with Ca2+ concentration. 5. A comparison of the rate of dissociation measured by fluorescence polarization and the rate of appearance of enzyme activity in the presence of a protein substrate suggests that the Factor XIII is autoactivated by a soluble a-subunit-containing molecular forming a tight complex with the substrate.


Molecular weights of the Thy-1 glycoproteins from rat thymus and brain in the presence and absence of deoxycholate. 1. The Thy-1 membrane glycoproteins from rat thymus and brain bound deoxycholate to 24% of their own weight as measured by equilibrium dialysis. The binding occurred co-operatively at the critical micelle concentration of deoxycholate, suggesting that the glycoproteins bind to a micelle, and not to the detergent monomer. 2. From sedimentation-equilibrium and deoxycholate-binding data the molecular weights of the glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoproteins respectively. The molecular weight of the polypeptide part of the glycoprotein is thus 12500. 3. In the absence of deoxycholate, brain or thymus Thy-1 glycoprotein formed large homogeneous complexes of mol. wt. 270000 or 300000 respectively. The sedimentation coefficient of these was 12.8 S. The complex was only partially dissociated by 4M-guanidinium chloride. 4. After cleavage of brain or thymus Thy-1 glycoprotein with CNBr, two peptides were clearly identified. They were linked by disulphide bonds and both contained carbohydrate. This cleavage suggests there is only one methionine residue per molecule, which is consistent with the above molecular weights and the known amino acid composition.


Kinetics and template-dependency of ribonucleic acid synthesis by bacterial ribonucleic acid polymerase. The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.


The pyridoxal-binding site in pyridoxamine-pyruvate transaminase. The enzyme-substrate complex formed between pyridoxamine-pyruvate transaminase (EC 2.6.1.30) and pyridoxal was reduced with NaBH4. After carboxymethylation and tryptic digestion, pyridoxyl-lysine-containing peptides were isolated by a combination of Sephadex and Dowex 50 chromatography. Analysis of these peptides shows the structure around the pyridoxal-binding lysine residues to be Ala-Asp-Ile-Tyr-Val-Thr-Gly-Pro-Asx-Lys(Pxy)-Cys-Leu(Pro2, Gly2, Ala2, Met)(Thr, Leu2)Gly-Val-Ser-Glu-Arg. This structure differs from those found for the corresponding peptides from pyridoxal phosphate-dependent enzymes.


A theoretical analysis of the use of zonal gel filtration in the detection and purification of protein-ligand complexes. A simple model of zonal gel filtration is analysed numerically to establish semiquantitatively how the capacity of a protein-ligand complex to survive gel filtration intact depends on several experimental variables and on the kinetic characteristics of the complex. The design of gel-filtration experiments aimed at detecting and partially purifying binding proteins is discussed.


Physical measurements of the liver glucocorticoid receptor. Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 X 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.


Phosphorus nuclear-magnetic-resonance studies of compartmentation in muscle. 1. Phosphorus nuclear-magnetic-resonance measurements were made on rat vastus lateralis muscle. 2. In the absence of oxygenation, the resonance from P1 broadens, as the 'energy pool' of the muscle gradually runs down. This, together with measurements of transverse relaxation times (T2) indicate that the intracellular pH is not uniform within the muscle volume. 3. Incubation of the muscle with acetate buffer at low pH (5.2) results in splitting of the P1 resonance into two components; one corresponds to phosphate in a low-pH environment and the other to phosphate in its original environment. These observations indicate that P1 is distributed among different compartments in the muscle cell. 4. Compartmentation of sugar phosphate (mainly glucose 6-phosphate) is also indicated by this method, but no evidence has been obtained for this type of compartmentation of ATP and phosphocreatine.


Testosterone regulates the synthesis of major proteins in rat ventral prostate. The presence of three major proteins alpha, beta and gamma in rat ventral prostate was demonstrated by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Their regulation by androgens was studied by measuring the rates of synthesis of the proteins in minced prostatic tissue by using L-[35S]methionine. The three proteins account for 30-40% of the proteins synthesized in the gland. After castration, their rates of synthesis rapidly decline to about 1% that of normal animals, and this cannot be accounted for by the accompanying decrease in general protein synthesis. Testosterone reverses these changes in castrated animals, so that after 4 days normal synthesis is restored. The regulation is specific for androgens, since cyproterone acetate, an anti-androgen, is inhibitory and oestradiol-17beta and corticosterone are without effect. Preliminary characterization of the proteins indicates that protein alpha (mol.wt. 22000, pI unknown) is a glycoprotein containing glucose and/or mannose residues and occurs in both the mitochondrial and cytosol fractions. Protein beta (mol.wt. 12000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also found exclusively in the cytosol fraction.


Ornithine decarboxylase activity and the onset of deoxyribonucleic acid synthesis in regenerating liver. Compared with normally fed animals, rats fed on a low-protein diet for 3 days exhibit a considerable delay in DNA synthesis after partial hepatectomy. In the regenerating livers of these animals (a) the timing of the first peak of ornithine decarboxylase activity is not altered and (b) the second peak of enzyme activity is delayed by a few hours, but polyamine concentrations are similar to those of normally fed rats. The results suggest that regardless of the possible effect of polyamines on DNA synthesis, the time course of ornithine decarboxylase activity appears to be independent of the onset of DNA replication in regenerating livers.


The effect of copper excess on iron metabolism in sheep. Sheep were treated with large amounts of copper (20 mg of CuSO4,5H2O/kg body wt. per day) for 9 weeks to examine the effect of copper excess on iron metabolism. In addition to confirming that massive haemolysis and accumulation of copper occurs in the liver, kidney and plasma after 7 weeks of exposure to excess copper, it was observed that excess copper produced an increased plasma iron concentration and transferrin saturation within 1 week. Further, iron preferentially accumulated in the spleen between 4 and 6 weeks of copper treatment, producing 3-fold increases in the iron content of both the ferritin and non-ferritin fractions. A 3-4 fold increase was also observed in the amount of ferritin that could be isolated from the spleen. The copper treatment had little or no effect on the concentration of iron in the liver and bone marrow. The following properties of erythrocytes were also unaffected by copper treatment: size, haemoglobin content and pyruvate kinase activity, although the erythrocyte concentration of copper increased after 6 weeks. Copper accumulated in the spleen between 6 and 9 weeks, probably owing to the phagocytosis of erythrocytes containing high concentrations of copper. The data suggest that copper excess influences iron metabolism, initially by causing a compensated haemolytic anaemia, and later by interfering with re-utilization of iron from ferritin in the reticuloendothelial cells of the spleen.


Studies on the mitochondrially bound form of rat brain creatine kinase. 1. The development of the total rat brain creatine kinase was studied in brain homogenates. Until approx. 14-15 days after birth, the activity remains less than one-third that of the adult activity (207+/-6 units/g wet wt. s.d.; n=3). Over the next 10 days the activity increases markedly to the adult value and thereafter remains essentially constant. 2. In the adult brain, approx. 5% (11.9+/-2.2 units/g wet wt. s.d.; n=5) of the total creatine kinase is associated with the mitochondrial fraction. This creatine kinase could not be solubilized by sodium acetate solutions of up to 0.8m concentration, whereas 66% of the hexokinase associated with brain mitochondria was released under these conditions. 3. Rat brain mitochondria incubated in the presence of various concentrations of creatine (1, 5 and 10mm) and ADP (100mum) synthesized phosphocreatine at rates of approx. 4.5, 11 and 17.5nmol/min per mg of mitochondrial protein. Atractyloside (50mum) or oligomycin (1.5mug/mg of mitochondrial protein) completely inhibited the synthesis of phosphocreatine. 4. The apparent K(m) and V(max.) values of the mitochondrially bound rat brain creatine kinase were determined in both directions. The V(max.) in the direction of phosphocreatine synthesis is 237nmol/min per mg of mitochondrial protein, with an apparent K(m) for creatine of 1.67mm and for MgATP(2-) of 0.1mm, and in the reverse direction V(max.) is 489nmol/min per mg of mitochondrial protein, with an apparent K(m) for phosphocreatine of 0.4mm and for MgADP(-) of 27mum. 5. The results are discussed with reference to the role that the mitochondrially bound creatine kinase may play in the development of brain energy metabolism.


Lipoprotein lipase activity in the tissues of guinea pigs exposed to different dietary fats from conception to three months of age. Long-term exposure of guinea pigs to a diet rich in maize oil caused an increase in adipose-tissue lipoprotein lipase activity. A similar diet rich in beef tallow had no such effect, and neither diet affected the enzyme activity in heart, lung, diaphragm or skeletal muscle.


The metabolic fate of glutamine nitrogen in the perfused rat kidney. The fate of glutamine amide and amino nitrogen was determined in the perfused rat kidney. There is a deficit of about 40% in the recovery of glutamine nitrogen and NH3 in the perfusion medium. After correction for the tissue conetent of glutamine, the remaining nitrogen was fully accounted for by small amounts of NH3, glutamate, aspartate and a compound that liberated glutamate on acid hydrolysis, probably 5-oxopyrrolidine-2-carboxylate (pyroglutamate). The latter accounted for 8% of the glutamine metabolized.


The pentose phosphate pathway in regenerating skeletal muscle. 1. The activities of the oxidative enzymes (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and of the non-oxidative enzymes (transaldolase, tranketolase, ribose 5 phosphate isomerase and ribulose 5-phosphate 3-epimerase) of the pentose phosphate pathway were measured at various times during the first 24h of skeletal-muscle regeneration after administration of Marcaine, a mytoxic local anesthetic. 2. The activities of the oxidative enzymes increased after Marcaine injection and rose to 9 times control activities by 24h. 3. The activities of all non-oxidative enzymes were increased after Marcaine administration, but to a much smaller extent than the oxidative enzymes (1.1-1.7-fold). 4. Histochemical analysis localized glucose 6-phosphate dehydrogenase activity within muscle fibres of control and Marcaine-treated muscles. 5. Cycloheximide or actinomycin D prevented the increase in oxidative enzyme activities, suggesting a requirement for synthesis of protein and RNA.


Oestriol-stimulated synthesis of ribonucleic acid in the uterus of the immature rat. Oestradiol-17beta and oestriol both sequentially stimulate the synthesis of heterogeneous nuclear RNA and rRNA in vivo. However, the stimulation by oestradiol-17beta, particularly of rRNA synthesis, is very much greater than that elicited by oestriol.


Surface glycosaminoglycans and calcium distribution in 3T3 cells. Resting 3T3 cells have relatively more sulphated glycosaminoglycans and Ca2+ in their cell coat than do growing or SV40-virus-transformed cells. It is suggested that the different Ca2+-binding capacity controls cellular activities by affecting the distribution of Ca2+ between the intra- and ecto-cellular compartments.


Synthesis of guinea-pig cardiac myosin as measured by constant infusion. An equation was derived from which the turnover time of individual muscle proteins could be calculated from measurements made at a single time interval in individual animals after initiation of constant intravenous infusion of labelled amino acid. The calculation requires only the specific radioactivities of the amino acid in plasma, in the intracellular fluid and in the protein under study. Pool sizes were not required. When the equation was applied to adult guinea-pig cardiac myosin, the average turnover time was 16 +/- 1 days.


Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis. 1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.


Transport of phosphoenolpyruvate through the erythrocyte membrane. Phosphoenolpyruvate was transported through the erythrocyte membrane at low pH (4.5-6.5). The influx was observed not only in an iso-osmotic sucrose medium, but also in 0.1 M-citrate solution, but it was negligible in an iso-osmotic NaC1 solution. Efflux, however, was observed in both the sucrose and NaC1 solutions. Compounds derived from phosphoenolpyruvate by replacing the methene group by similarly hydrophobic groups such as hydrogen or the methyl group were permeant but those with the hydrophilic hydroxymethyl group were impermeant. This transport was inhibited by the treatment with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid or pyridoxal phosphate/NaBH4, which are known to be specific for the transport of anions such as C1-, SO42- and HPO42-. It showed saturation kinetics with respect to phosphoenolpyruvate concentration in the medium. These results suggest that the transport of phosphoenolpyruvate is mediated by the anion-transport system. Although phosphoenolpyruvate was transported against the concentration gradient, the transport was characterized as a passive transport, and this apparent uphill transport was interpreted by the Donnan equilibrium.


Comparison of the metabolism of chylomicrons and chylomicron remnants by the perfused liver. 1. The hepatic metabolism of chylomicrons and chylomicron remnants was compared after adding approximately equal numbers of each lipoprotein particle to the perfusate of isolated livers. 2. At least 40% of the added remnants were metabolized by the liver compared with less than 3% for chylomicrons. 3. There was significantly more net removal of labelled remnants than of chylomicrons by the liver. 4. A greater proportion of labelled cholesterol than of labelled triacylglycerol fatty acids was transferred to the liver from each lipoprotein. 5. Cholesteryl esters of remnants were hydrolysed to triacylglycerol fatty lipoprotein. 5. Cholesteryl esters of remnants were hydrolysed to triacylglycerol fatty acids of remnants were oxidized to CO2 more extensively than those of chylomicrons. 6. There was greater oxidation of remnant glycerolipic [(1(-14)C]oleate than of glycerolipid [1(-14)C]palmitate. 7. A large fraction of the fatty acids of remnants, but not of chylomicrons, was transferred to phospholipids, which were released by the liver in a lipoprotein of relative density less than 1.006. 8. Label from remnants, but not from chylomicrons, was found in lipoproteins of relative density greater than 1.006, which were not released during perfusion but could be flushed out from the liver at the end of perfusion.


The mode of action of alpha sarcin and a novel assay of the puromycin reaction. A new technique was developed for measuring the amount of peptidyl-tRNA in a protein-synthesizing system in vitro. By this technique the course of the puromycin reaction may be followed and the modes of action of various inhibitors of protein synthesis readily determined. We conclude that the polypeptide alpha sarcin inhibits the binding of aminoacyl-tRNA into the ribosomal 'A' site, that sparsomycin inhibits the peptidyl transferase reaction and that cycloheximide may block translocation.


Changes in the response of mitochondrial calcium transport to exogenous phosphate during development in flight muscle of the sheep blowfly Lucilla cuprina. 1. Ca2+ transport by mitochondria isolated from flight muscle of the sheep blowfly Lucilla cuprina does not occur in the absence of added P1. Maximum rates of transport are attained when about 2.5 mM-phosphate is present. 2. As mitochondria develop, high but not low phosphate concentrations begin to inhibit Ca2+ transport markedly; those isolated from 2-day-old flies for example, are inhibited by about 75% by 20 mM-phosphate. Maximum rates of transport, i.e. those measured in the presence of 2.5 mM-phosphate, begin to decline only when the fly is about 3 days old. 3. Mitochondrial phosphate transport activity does not change during development of the blowfly, but the endogenous concentration of the anion does. At emergence it is about 6nmol/mg of protein, increases to about 17 nmol/mg of protein at 2-3h and then rapidly declines to reach less than 5 nmol/mg of protein after 2 days of adult life. 4. Studies on the effect of phosphate on oxidation of alpha-glycerophosphate in the absence and presence of ADP reveal a lack of inhibition by high phosphate concentrations indicating that the anion does not influence Ca2+ transport by preventing the generation of the proton electrochemical gradient across the inner membrane. 5. It is concluded that the molecular assembly in the inner membrane of Lucilla mitochondria responsible for transporting Ca2+ is fully developed at emergence and remains so for at least 2-3 days of adult life. The possibility exists that Ca2+-transport activity in these mitochondria is controlled at least in part by P1.


Properties of energy-dependent calcium transport by rat liver microsomal fraction as revealed by initial-rate measurements. Measurements of the initial rate of Ca2+ transport by rat liver microsomal preparations reveal the existence of two phases of transport activity. The first, a phase of rapid transport, is complete by 3-5 min, at which time the second (slower) phase begins; this remains linear for up to at least 40 min. The initial phase is minimal in the absence of MgATP. The initial rate of Ca2+ transport reaches values as high as 25 nmol/min per mg of protein; the Km for Ca2+total is 1-2 micrometer and that for MgATPtotal about 500 micrometer. Ruthenium Red (3-5 nmol/mg of protein) has little effect on the initial rate of transport, whereas tributylin (2 micrometer) inhibits equally in a KC1- or a KNO3-containing medium. Compunds that  collapse components of the proton electrochemical gradient in mitochondria (valinomycin and carbonyl cyanide m-chlorophenylhydrazone) each inhibit by 70-80% the initial rate of microsomal Ca2+ transport.


Plasma lipids, triglyceride/fatty acid pattern, and plasma insulin in fasted healthy volunteers during continuous ingestion of ethanol. Influence of lipolysis inhibited by nicotinic acid. Healthy fasted volunteers were subjected to an acute oral ethanol load over 12 h after a diet of 3 days with high linolenic acid content. Free fatty acids, triglycerides, glycerol, phospholipids, cholesterol and insulin, as well as the fatty acid pattern of triglycerides in the plasma, were determined during the test. The test was repeated with nicotinic acid added. The lipid values obtained and the comparisons of fatty acid composition both indicate that the predominant role of peripheral lipolysis in the genesis of acute ethanol-induced hypertriglyceridemia, in spite of the possibility of enhanced synthesis of palmitic acid in the liver.


Fractional fatty acid incorporation into human adipose tissue (FIAT) in hypertriglyceridemia. Two processes regulate the removal of lipoprotein triglycerides from plasma: (1) hydrolysis of triglycerides by lipoprotein lipase to fatty acids in the capillaries; (2) local uptake and esterification of fatty acids by adipose tissue, muscle and other tissues. This second process has been studied on needle biopsy specimens of adipose tissue from normotriglyceridemic and hypertriglyceridemic subjects. Hypertriglyceridemie was found to be associated to low fatty acid incorporation into adipose tissue.


Platelet aggregation in patients with peripheral vascular disease. Platelet aggregation has been studied in 28 control subjects and 46 patients with peripheral vascular disease. The platelets from the patients with peripheral vascular disease were significantly more sensitive to the aggregating effects of adenosine diphosphate and adrenaline than those from the control group  (P less than 0.001). In addition, spontaneous platelet aggregation was observed in vitro in several of the vascular patients; this response was not apparent in the control group. Increased platelet aggregation could add to the vascular impairment in the unoperated subject and might also play a part in premature graft failure. Platelet function studies should perhaps be included as a routine in the assessment of patients with peripheral vascular disease.


Cholesterolemia and cardiovascular abnormalities in rats caused by copper deficiency. The association of copper with cardiovascular disease and a possible involvement of copper in the metabolism of cholesterol prompted the study on hypercholesterolemia mediated by copper deficiency. Copper deficient rats were found to exhibit a highly significant cholesterolemia (P less than 0.001), and plasma cholesterol showed a significant correlation with hepatic copper concentration (P less than 0.03). Two copper deficient rats died with hemothorax. The hearts of copper deficient rats were hypertrophied with large areas of hemorrhage, inflammation and focal necrosis. Prominent subendocardial fibroplasia was evident in copper deficient animals. The myocardial arteries of copper deficient rats were normal, however, aortas showed large areas of distorted and depleted elastic fibers. The results are discussed in terms of a possible role for copper in cholesterol metabolism, and in the pathogenesis of atherosclerosis.


Preparation of [14C]colestipol hydrochloride and its disposition in the human, dog and rat. Colestipol hydrochloride, a polymeric, ion-exchange type, hypocholesterolemic agent, acting by sequestering bile acids, was labeled with carbon-14. The disposition of the labeled material was studied in the human, dog and rat. The extent of absorption from the gastrointestinal tract, as judged by urinary excretion of radioactivity, was very small and correlated well with the contents of water-soluble and dialyzable materials in the colestipol hydrochloride. Results were consistent with the dialyzable material in the drug being the absorbable species.


Effects of acidic glycosaminoglycans in human aortic inner and outer layers on partial thromboplastin time. (1) Acidic glycosaminoglycans (AGAG) were prepared from the inner and outer layers of human aorta and fractionated by Dowex 1-X2 columns. The anticoagulant activity of the fractionated AGAG was measured by the partial thromboplastin time. (2) Heparan sulfates were the main AGAG in the 1.25 M NaCl fraction and were more abundant in the outer layer than in the inner. Likewise, there was more dermatan sulfate in the outer layer. (3) The AGAG in the outer layer showed much greater anticoagulant activity than those in the inner layer, both in the 1.25 M and 1.75 M NaCl fractions. (4) Anticoagulant activity was attributed to the heparan sulfates in the 1.25 M fraction and to the dermatan sulfate in the 1.75 M fraction.


[Erythrocyte potassium in premature infants small for gestational age]. A direct method was used to study the concentration of intraerythrocytic sodium and postassium in 21 prematures with adequate weight for their gestational age and in 24 small for their gestational age. Intraerythrocytic potassium showed to be less, while sodium turned out to be greater in the group of prematures small for their gestational age when compared with those with adequates weight. No significant differences were found between concentrations of serum sodium and potassium in both groups. These results are in close relationship with the findings of potassium in the muscular cell and in the leukocytes of infants with protein-calorie malnutrition and may show importance in terms of the flow of ions through the erythrocytic membrane.


[Atmospheric pollution and spirometry in healthy children of the Distrito Federal]. Investigation of the vital capacity, maximum expiratory flow and maximum mean expiratory flow was carried out in children living in two areas of Mexico City, one of them with a high degree (azcapotzalco) and the other one with low degree (Tizapán) of air pollution. Timed spirometry was carried out on children attending 4th, 5th and 6th grades in two official grammar schools so both groups were as uniform as possible in their social-economical level and nutritional and health conditions. This goal was attained quite satisfactorily and groups turned similar, after comparing results and analysing statistically somatometric data, together with scholarship of the parents, economical income, history of respiratory diseases and physical examination. However, quite a significant statistical difference (p less than 0.001) was found between the parameters involving measurements of ventilation in favor of Tizapán children. The difference between Azcapotzalco and Tizapán children in terms of the three ventilatory parameters analyzed was possibly due to the fact the former lived in a greater polluted area.


[Analysis of 2 studies of weight and height separated by a 50-year interval in children of Mexico City]. The investigation of somatometric values to be used as reference patterns for Mexican children has de be scanty; the author mentions the most significant works carried out within the last 50 years. Considering that the material is comparable and in view of the magnitude of the samples, (4,757 vs. 5,533 cases) the author analyses the values published in 1926 and his own (1975). Differences in weight, height and corporal surface were found in both sexes at 5 years, which gradually increases to become quite marked at 17 years of age. At the same time, there was acceleration in development which was calculated in 14 months within 50 years; thus, in 1926, menarche was expected at 159 months of age when the average weight reached 45,600 gms. and height was 149.5 cm., while at present, it is expected at 144 months, with same weight and height. The rate weight/height has remained unchanged, but the silhouette has changed because of greater relative growth of the lower segment. The author comments that these changes, favorable to the privileged groups may not be found in the rural areas and that in some places, the height in females is gradually decreasing at the rate of 1 cm, every 5 years, which means a remarkable impairment.


[Dermatomyositis in pediatrics].

[Dermatomyositis in pediatrics]. The study included 19 children with dermatomyositis. All showed frequently atypical cutaneous disords which in most cases were the initial manifestation. In every case there was some clinical evidence of proximal myopathy. Arthralgias were frequently found and sinusal tachycardia was present in 10 cases. The correct diagnosis was made from the beginning only in 6 cases. Systemic lupus erythematosus was the main cause of confusion. The differential diagnosis between these two entities cannot be made only by the presence or not of nephropathy, even if this disease was evident in 42% of patients with dermatomyositis. The most useful laboratory tests for the diagnosis and management were CPK, aldose and serum TGO. Electromyography was abnormal in every case where it was practiced. Muscular biopsy was valuable in 15 of the 19 patients. All were treated with prednisone and in most cases with good results.


[Obstructive vascular ring. Report of a case]. The case of an infant with obstructive vascular ring is reported. Considerations are made on the most frequent types of this anomaly, its embryology, clinical picture and its diagnosis and taking into account advances made of the surgical treatment in recent years, the importance of the diagnosis is emphasized together with the consequence of complications in untreated cases.


[Reye's syndrome: pathological aspects]. A clinical and statistical review of Reye's syndrome is made, reporting the case of a female seven months old infant with disturbances of the digestive tract associated to hepatomegaly, convulsive crises, maculopapular exanthema, extreme hypoglycemia, hyperammoniemia and high blood urea. The post-mortem study showed fine drop diffuse liver steatosis, renal tubuli steatosis, focal steatosis of myocardial fibers, generalized meningoencephalic edema and viral pneumonia. These clinico-pathologic characteristics lead to the diagnosis of Reye's syndrome, an infrequent entity in our milieu judged by national literature.


[Intestinal atresia].

[Intestinal atresia]. The authors reviewed their experience with 20 cases of intestinal atresia (jejunum, ileum and colon) admitted between January 1972 and January 1977. International literature shows 80% mortality rate for this malformation before 1940 and 10-25% in current reports. In Mexico, before 1959, the mortality rate was 80%, but the most recent reports showed 28% mortality rate for atresia and 33% in cases of stenosis. The group of patient studied included twelve cases with a weight over 2,500 g. and eight patients under 2,500 g. Four patients showed multiple associated malformations (incompatible with life in one case) and in 17, there were no associated malformations. Nineteen cases were operated, the dilated and atresic segments were resected and anastomosis was performed. It was termino-terminal in 13 cases and termino-lateral in 6. One case was explored surgically and no attempt to repair the atresias was made, because there were malformations incompatible with life. Four patients died. Even if this series is very small, the associated malformations, low weight and techniques used do not seem to influence the prognosis of these patients.


[Atresia of the esophagus. Experience in 33 patients]. The authors present their experience in the management of 33 patients with esophageal atresia. Twenty-six of them were type III (fistula from the lower end of the esophagus); four cases were "H" type of fistula and three cases were type one (with no fistula). In the type III group (26 patients) the major associated malformations seemed to be the most important factor that contributted to the mortality rate. Birth weight and the age at the moment of the diagnosis, did not seem to influence the final outcome. In this group, 13 patients died (50% mortality), ten patients had major associated malformations, all of them died (100% mortality). 16 patients did not have associated malformations; from them, only three died (18% mortality). All the patients with type "H" malformations survived. All the patients with type I malformations underwent gastrostomy and a cervical esophagostomy. One patient died in the immediate postoperatory period. The second one underwent a successful colon interposition and the third one was lost from control.


[Globular sedimentation rate as an index of infection in the newborn]. Sedimentation (VSG) rate was studied following the microhematocrit technique in 40 healthy and in 52 infected preterm infants complaining of gastroenteritis, bronchopneumonia, meningitis and septicemia. In healthy infants, VSG was measured at 3, 6, 9, 15, 20, 25 and 30 days of age and in infected cases, it was measured in the course of disease or during recovery. Increment of VSG was shown in healthy cases from 1.29 mm/1 hour at the age of 3 days up to 9.39 mm/1 hour at one month of age. In infected newborns, average VSG was higher than in healthy infants in every one of the days measured. In 8 of the infants with septicemia, VSG showed a marked rise followed by drop as the patients improved. At the end, comments are made on the usefulness of the method as and aid in the diagnosis of infection in preterm newborns.


[Furosemide at high doses in a girl with chronic renal failure]. A case is reported of a female preschool, age child with nephrotic syndrome, probably secondary to focal and segmentary hyalinosis with progressive decrease of the renal function. The patient was given increasing doses of furosemide to control the important retention of fluids she showed until a daily dose of 120 mg/kg. of body weight was reached. Adequate control of edema was managed and it was possible to decrease sodium restriction from the diet when urinary excretion of sodium increased. Such effect was useful for four months during which, the patient's diet turned more adequate and there was improvement of the nutritional state which was seriously impaired because of the restrictive diet given. However, control was lost because the patient died at home from bronchopneumonia. Thus, it is considered furosemide may be used in chronic renal failure at higher than usual doses in selected patients.


[Anatomo-radiological correlation of shock lung in pediatrics]. We reviewed three post-mortem studies of pediatric patients who died with the diagnosis of shock lung. We try to correlate the thorax X Ray studies obtained during clinical shock, with the anatomopathological findings. We established the basis for the anatomopathological and radiological diagnosis, and its comparation with the adult studies. No correlation could be establish with this a prospective study to try recognize this entity during life with serial X Ray studies, like was performed in adults.


[Gaucher's disease. Morphological study of lymph nodes by electron microscopy]. The morphological study of lymph node biopsies by light and electron microscopes is reported. The patient was a 7 months old infant with Gaucher's disease and infection symptoms of upper respiratory ducts. We believe this to be a case of lesser affection, since the patient was admitted complaining of complications that gave way to medical treatment. Lymphadenopathy and moderate liver and splenomegaly were occasional findings during examination. Through the light microscope, Gaucher's cells showed an eccentric nucleous, cytoplasmic striae which, at the electron microscope showed to be tubular bodies.


Immunogenetics of Lennox-Gastaut syndrome: search for LD determinants as genetic markers of the syndrome. In a previous work we demonstrated a strong association between HLA-B7 and the susceptibility to the Lennox-Gastaut syndrome. In this work we report some preliminary results obtained performing one-way mixed lymphocyte cultures in vitro between 15 unrelated children showing the typical EEG and clinical patterns of the syndrome. The same children were also tested against unrelated control cells from healthy donors. The low stimulation displayed by Lennox-Gastaut patients between them as compared with the higher stimulation found in patients/controls and controls/controls comparisons suggests the existence of an LD allele which may be associated with the disease.


[Evaluation of the effectiveness of thiabendazole, pyrantel and mebendazole in the treatment of necatoriasis in children]. A study was made of 48 patients from the different departments of the Hospital del Niño, DIF, in which hookworm eggs were demonstrated by qualitative coproparasitoscopic study (CPS). During the study, quantitative CPS was practiced on them by Stoll's dilution and the Harada-Mori technique, for the identification of the hookworm larvae. They were divided into three groups: A, B and C (16 patients in each group); the study was single-blind. The effectiveness of the evaluated drugs was based on the disappearance or diminution of hookworms eggs in the CPS (Stoll) practiced 2 weeks after the drug treatment had finished. The work was done with patients parasitized by Necator americanus (verified by the Harada-Mori technique). The A group was given pyrantel pamoate; the B group, thiabendazole and the C group, mebendazole. The results obtained were statistically processed by means of X2 proportions. There was no significant difference between the three groups when a light to moderate necatoriasis was treated; when a massive infection was involved, there was a significant difference and it was found that the best drug was pyrantel pamoate administered at the dose of 20 mg/kg/day/3 days. There were few side effects in the three groups, but they were not sufficient to cause suspension of the treatment. The cheapest drug was thiabendazole.


Applications of the macrophage electrophoretic mobility test (M.E.M.). This paper describes the principle of the M.E.M. test (macrophage electrophoretic mobility). Human lymphocytes from normal or diseased patients, if exposed to an antigen to which they were sensitized may produce a factor which would reduce the electrophoretic mobility of a normal guinea pig peritoneal macrophage population. It is shown a summary of the applications of the test system and disadvantages of using P.E.C. and a summary of test system used for assessing slowing factors produced by lymphocyte-antigen reaction.


[Trichotillomania].

[Trichotillomania]. Trichotillomania is a relatively frequent cause of alopecia in children. An area of the scalp, of irregular shape, partially devoided of hair or eventually a geometrically designed alopecic area should suggest the diagnosis. The histologic alterationes are characteristic. Most children with trichotillomania have mild alterationes of personality, some require psychiatric evaluation.


E-UFA test in multiple sclerosis.

E-UFA test in multiple sclerosis. Field et al. have recently suggested a blood test for multiple sclerosis based on determination of the absolute electrophoretic mobility of erythrocytes alone and in the presence of linoleic acid (LA) or arachidonic acid (AA). We attempted to reinvestigate this phenomenon in an Italian population.


M.S. diagnostic test: a comparison of different electrophoretic and immunologic methods for CFS IgG estimation. In order to select the most discriminative laboratory method of MS diagnosis, the results obtained with CSF IgG/total protein ratio, CSF-IgG Index, agarose and cellulose acetate electrophoresis were compared. The electrophoretic methods appear to be more discriminative for MS than IgG/total protein ratio or the CSF-IgG Index. Furthermore, cellulose acetate electrophoresis was found to be simpler and quicker to perform than agarose electrophoresis.


Another possible precipitating factor in multiple sclerosis: the exposure to organic solvents. Recent clinical observations seem to indicate that a possible correlation may exist between the appearance of inflammatory diseases of the nervous system and exposure to organic solvents. In certain cases the patients could not be distinguished, either from the clinical or laboratory point of view, from typical cases of multiple sclerosis. These observations raise the problem of a possible action of organic solvents on the immunological responses, already described in different pathological conditions as a further possible precipitating factor in MS.


[Correct placement of the endotracheal tube]. The authors present their experience with correct placement of an endotracheal tube. Thirty children were intubated for different indications following the same method. The correct position of the tube was confirmed by chest films. The method included: 1. Tracheal tube introduction by direct laryngoscopy. 2. Deliberate tube displacement to the main right bronchus. 3. Auscultatory carina detection while giving positive pressure ventilation with ambu, when the respiratory murmur appears in the left hemithorax. 4. Tube withdrawal a distance equal to the patient's little finger length, leaving off the tracheal tube tip at an appropriate distance from the carina. The placement was found correct in 29 cases.


[Proctologic examination in pediatrics]. The study included 873 rectal examinations carried out in children where inspection, rectal examination and endoscopy were performed in each case. Most examinations were done in children complaining of gastroenterocolitis. Inspection of gluteal, perianal and sacrococcygeal areas was normal in most cases: however, ammoniacal erythema was frequently found (44.2%). Rectal examination showed hypotonicity of the external sphincter in 146 cases which was linked to prolonged diarrhea and malnutrition. At the endoscopy, the rectal mucosa showed ulcerations, congestion, friability, edema and bleeding; however, it was considered normal in 274 occasions. In 540 patients, the endoscopic diagnosis was colitis of different types. In 54 cases, the endoscopic examination, plus the examination of fecal mucus taken directly from the rectal mucosa led to the diagnosis of amebic colitis. In opposition with adults, neoplastic lesions were uncommon and usually corresponded to leukemic infiltration or tumoral metastases. There were no complications attributable to the process.


Chemical modifications of the protein of carcinoembryonic antigen: associated changes in immunological activity and conformation. Chemical substitution of the exposed residues of tryptophan, tyrosine, histidine and arginine in carcinoembryonic antigen (CEA), using appropriately selective reagents, caused no significant change in the capacity of the antigen to bind to anti-CEA serum. However, treatments of CEA with 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, both in the presence of guanidine HCl, caused a large reduction in binding capacity. Measurement of the circular dichroism spectra of all of the products showed that retention of conformation of the molecular correlated well with retained antigenic activity, whereas the large losses in capacity to bind to anti-CEA sera were accompanied by a probably the result of gross conformational changes. The tyrosine residues of CEA may be classified into three categories: (i) 3 freely reacting residues, (ii) 7 or 8 moderately buried residues and (iii) 15 unreactive residues.


Maintenance of biological and biochemical characteristics of human colorectal tumours during serial passage in immune-deprived mice. The effect of serial passage in immune-deprived mice on certain biological and biochemical parameters has been studied in a series of 6 human colorectal tumour xenografts. Histological integrity is maintained for up to 10 serial passages, together with production of epithelial mucins and carcinoembryonic antigen. Passaged tumours retain human lactate dehydrogenase and glucose-6-phosphate dehydrogenase isoenzyme patterns and a human chromosome constitution. The induction of a murine tumour has been identified in this system, and the importance of routine checks for the presence of human tissue during serial passage is stressed.


Growth characteristics of human colorectal tumours during serial passage in immune-deprived mice. The growth characteristics of 6 human colorectal tumours have been examined during serial passage in both male and female immune-deprived mice. Exponential growth is a characteristic feature, especially on very early passages. Growth rates in 5 out of the 6 tumour lines increase during the first few transplant generations. This is accompanied by a shorter exponential growth phase and an increased slope of the growth curves. Lag phases and growth rates for individual tumours are variable within a passage. Growth rates for tumours maintained within the same host are similar, and are at least partially influenced by the host. In one tumour line examined in detail, the increased growth rate is attributable to a decreased cell-loss factor, and the difference in growth rate between human colorectal tumours and their corresponding xenografts may therefore largely be due to a difference in the contribution of this factor.


Cytogenetics of malignant epithelial cells and lymphoblastoid cell lines from nasopharyngeal carcinoma. The malignant epithelial cells of nasopharyngeal carcinoma (NPC) and cells of lines derived form the lymphoid cells which infiltrate this tumour have been investigated cytogenetically. Chromosome spreads of lymphoblastoid cells of lines established from 7 different NPC biopsy specimens were examined after banding staining. Banding was also applied to the epithelial tumour cells of 5 further biopsy specimens freed from non-malignant infiltrating cells by passage through nude mice; epithelial cell spreads were obtained by in vivo splindle arrest. Five of the lymphoblastoid lines were found to be diploid, and 2 tetraploid; the karyotypes were essentially normal. The squamous epithelial nature of the cells in the nude-mouse-grown NPC tumours was established by light and electronmicroscopy, and 3 tumours were found to be near-triploid, and 2 near-diploid. The cells of the near-triploid tumours contained grossly abnormal chromosomes but those of the near-diploid tumours showed only relatively minor changes. Although abnormalities were observed which were specific for cells from each individual tumour, no discernible change was common to cells from all the tumours.


The structure of tumours derived from mouse submandibular gland epithelium transformed in vitro. The morphology and ultrastructure of 48 primary tumours established from 5 cell lines of adult mouse salivary gland epithelial cells transformed in vitro are described. Tumours from 4 of the cells lines were adenocarcinomas with a wide range of structural variation, and resembled human salivary gland carcinomas. The fifth cell line produced tumours with carcinomatous and sarcomatous elements.


Quantitative relationship between volume of tumour cell units and their intravascular survival. The derivation of the median volume (MV) and the geometric standard deviation (SDg) for a suspension of tumour cells quantifies the size and distribution of tumour cell aggregates in the suspension. Data collected in a group of 14 experiments shwoed a significant correlation of 0.80 (P less than 0.001) between the number of lung tumours formed by a suspension of B16 melanoma cells injected i.v. into C57BL/6J mice and the product of the MV and SDg of each cell suspension. These data define a size parameter of tumour cell suspensions that correlates with the intravascular survival properties of tumour cells.


Antitumour responses induced by short-term pretreatment with tumour cells. The injection (s.c. or i.p.) of 10(6) live or lethally irradiated methylcholanthrene-induced fibrosarcoma cells into CBA/Ca mice one or 2 days before i.v. challenge with the same tumour inhibited the formation of artificial lung tumour metastases. In addition, it also frequently enhanced the cytostatic effect of peritoneal exudate cells on monolayers of the same tumour. The effects on lung tumour metastasis were not noted if X-irradiated tumour was injected i.v., or if s.c. administration was delayed until one day after i.v. challenge. Similar effects on tumour growth were also observed in C3Hf/Bu mice and (CBA/Ca x A/HeJ) F1 hybrids which were pretreated (s.c.) with tumour shortly before i.v. challenge with the same tumour. Further studies in CBA/Ca mice suggested that the protective effect was tumour-specific, for the growth of i.v. injected tumour was not significantly inhibited by pretreatement with a number of other MC-induced or spontaneous tumours from the same and different strains.


Familial breast cancer: report of a family pedigree. Following a report of several relatives suffering from breast cancer, the occurrence of neoplasms in 3 generations of a large family was carefully checked. Members of one out of 8 branches were found to have a high incidence of breast cancer with 6 women affected, 4 of them under the age of 40. As well as early onset, these women presented other features typical of "breast cancer families": bilateral breast cancer, other second primary tumours, ovarian cancer in the daughter of one affected patient, and benign breast disease in the sister of another.


Controlled study of PUVA and adjunctive topical therapy in the management of psoriasis. In a random study of 116 patients with psoriasis vulgaris, oral psoralen photochemotherapy (PUVA) used alone was compared to PUVA plus adjunctive topical therapy with tar, dithranol, or topical corticosteroids. PUVA plus topical corticosteroids produced more rapid clearing of psoriasis but despite maintenance therapy, the frequency of recurrences of the disease during the early phase of follow-up was significantly higher with that treatment, as compared to all other treatments. Dithranol plus PUVA also cleared psoriasis quicker than PUVA alone but patient acceptability for that regimen was low. The addition of tar to PUVA therapy appeared to have little influence on results.


Dose-effects of 8-methoxypsoralen and UVA in cultured human lymphocytes. Cultures of human lymphocytes were incubated with 8-methoxypsoralen (8-MOP) and irradiated with long wave ultraviolet light (UVA). Subsequently the mitogenic response to PHA was determined by 3H-thymidine incorporation. The minimal effective dosages of drug and light for the partial suppression of lymphocyte PHA-responsiveness were found to be 0.1 microgram/ml 8-MOP and 0.5 J/cm2 UVA. While an increase in the concentration of 8-MOP revealed a comparatively small rise in phototoxicity, a marked inhibition of 3H-thymidine uptake was obtained by increasing the UVA dosage. Cell viability of non-stimulated lymphocytes was similarly affected by the combined treatment.


Psoriasis and stress. II.

Psoriasis and stress. II. A retrospective assessment of insight was undertaken in 61 patients whose psoriasis was considered to have been precipitated by stress. A significantly better prognosis was observed in those patients assessed as having had insight.


Precursor keratin protein from human epidermis. Prekeratin has been isolated from human epidermis using the method of Matoltsy for bovine nose epidermis. Analysis by sedimentation equilibrium indicates that human prekeratin is heterogeneous in solution. The molecular weight range is from about 200 X 10(3) to at least 2 X 10(6). Polyacrylamide gel electrophoresis shows three polypeptide chains of molecular weights 65,000 60,000 and 58,000. A lower than average ratio of polar/apolar amino acid residues suggests that hydrophobic interactions are important for stability.


Low nickel diet in the treatment of patients with chronic nickel dermatitis. Of 28 patients with chronic nickel dermatitis 17 experienced aggravation following oral ingestion of 2.5 mg nickel, but not a placebo tablet. The dermatitis of 9 of the 17 patients improved during a period of 6 weeks on a low nickel diet. The dermatitis of 7 of the 9 patients flared again when a normal diet was resumed. Nickel excretion over a 24-h period was measured for 14 of the 17 patients by atomic absorption spectrophotometry before, during, and after the diet. Reduced excretion was seen during the diet with no statistically significant difference between patients whose dermatitis improved during the diet and those whose dermatitis showed no change.


Treatment of eczema with cyclophosphamide and azathioprine. Nine patients with eczema were treated with the immunosuppressive drugs cyclophosphamide and azathioprine. In all patients the eczema was severe and incapacitating and had not responded satisfactorily to systemic and topical corticosteroid therapy. The eczema improved in all patients and systemic corticosteroids, which had resulted in undesirable side-effects, could be decreased or stopped. Leukopenia was not necessary for achieving clinical improvement. Several months of treatment were needed to obtain significant improvement in the eczema and long term remissions followed cessation of therapy.


An animal model for evaluating the local and systemic effects of topically applied corticosteroids on epidermal DNA synthesis. A simple animal model for studying topically applied glucocorticosteroids for their effect on epidermal DNA synthesis is described. It is designed so that their potential for systemic side effects may be predicted and its usefulness in this respect is illustrated by the results obtained with two of the steroids investigated. One, a new formulation Temetex, is predicted to have considerable systemic potential. The other, Molivate, is remarkable for having moderate local, without demonstrable systemic activity.


Photosensitivity after treatment of porphyria cutanea tarda with low dose chloroquine. Treatment of a patient with porphyria cutanea tarda with low dose chloroquine improved the clinical and biochemical abnormalities in association with a reduction in photosensitivity as shown by changes in the action spectrum.


Opportunistic infection of chronic skin ulcers with Pseudomonas putrefaciens. Heavy growth of Pseudomonas putrefaciens was isolated repeatedly in mixed culture with other Gram-negative rods from chronic ulcers on the extremities of an elderly patient with burnt-out leprosy. Treatment was with systemic cotrimoxazole, topical framycetin and general supportive therapy, and the ulcers gradually healed over a period of 4 weeks.


Leg ulcers in alpha-thalassaemia (haemoglobin H disease). A case of recurrent leg ulceration associated with alpha-thalassaemia (haemoglobin H disease) is reported. It is suggested that leg ulcers occurring in the thalassaemic syndromes may be due to structural changes in the affected red cells which result in increased cell rigidity, decreased deformability and consequent diminished blood flow in capillary beds that are subjected to venous stasis.


Pathogenicity of the intercellular antibodies of pemphigus and their periodic removal from the circulation by plasmapheresis. The authors are convinced that the intercellular antibodies of pemphigus are pathogenic. Consequently periodic plasmapheresis in two patients with pemphigus has been performed to remove the antibodies from the circulation. After larger plasma-exchanges there was a decrease of the antibody titre and a parallel improvement of the clinical condition.


In vivo observations of skin capillaries in workers exposed to vinyl chloride. An English-American comparison. An unselected sample of vinyl chloride (VC)-exposed individuals, all employees of a polyvinyl chloride production plant in England (129 subjects), were examined by in vivo capillary microscopy. Results were compared with those previously obtained from a similar study in the USA to determine whether an association between VC exposure and capillary abnormalities could be found in different environments. The results showed a similar distribution of capillary abnormalities among the VC workers in both countries (39.5% in England compared with 36% in the USA). These capillary abnormalities were significantly more frequent among VC-exposed subjects than among control industrial workers (7.7%, 6%). In vivo microscopy also showed a higher incidence of papular lesions (13.5%) than had been detected clinically (1%).


Respiratory function and symptoms in workers exposed simultaneously to jute and hemp. The environment and health of a working population exposed simultaneously to jute and hemp were studied. Classical symptoms of byssinosis were not present but 21 workers (7%) complained of atypical tightness of the chest. The prevalence of chronic bronchitis among the exposed workers was statistically significant in comparison with controls. Effects of dust concentrations, age and duration of exposure on the prevalence of chronic bronchitis were studied. A statistically significant reduction in FEV1.0 at the end of a work shift occurred in all the exposed workers. Bronchodilators given after the shift showed that acute reductions in forced expiratory volumes were nearly fully reversible in all exposed workers. Smokers and those with chronic bronchitis had greater reductions in FEV1.0 values at the end of the work shift.


Trichloroethylene exposure. Biological monitoring by breath and urine analyses. A mathematical model developed previously has been used to study some aspects of biological monitoring of exposure to trichloroethylene (TRI) by the analysis of this solvent in alveolar air or of its metabolites, trichloroethanol (TCE) and trichloroacetic acid (TCA), in urine. Assuming that a biological control must be representative of the time-weighted average concentration (TWA), it was found that sampling for TRI and TCE analyses must be carried out the morning after the exposure being considered. On the other hand, for a TCA analysis, the timing of urine sampling is not a determinant factor. Theortical limit concentrations have been set up for these biological indicators, but it is shown that their application must be restricted to exposures which are quantitatively reproducible from one day to the next. In all other cases, it appears that this monitoring method can lead to errors in the estimated exposure concentrations. A tentative method of biological monitoring is therefore proposed. It is based on the analysis of TCE in the urine or TRI in the alveolar air before and after the exposure being monitored. TCA is not considered to be sensitive enough to variations in the inspired concentration to be used as an indicator of a single exposure risk.


Differences following skin or inhalation exposure in the absorption and excretion kinetics of trichloroethylene and toluene. The concentrations of trichloroethylene in breath and blood and the urinary excretion of its metabolites following 30 minutes' direct immersion of one hand in the liquid, were compared with those obtained after four hours' inhalation exposure to the vapour of 100 ppm, described in a previous paper. The comparison shows that the end-tidal air concentrations during the first two hours of the post-exposure period were about twice as high in the case of skin exposure as in that of inhalation exposure, although the uptake of the solvent through the skin was only about one-third of the inhaled uptake. A kinetic approach suggested that differences in trichloroethylene movement in the body would be a principal cause of this discrepancy. The results of a similar series of experiments using toluene suggested that it is less readily taken up than trichloroethylene through the skin. It was concluded from the present investigation that analyses of not only breath but also of blood or urine are necessary and toluene would rarely be absorbed through the skin in toxic quantities during normal industrial use.


Absorption of m-xylene vapours through the respiratory tract and excretion of m-methylhippuric acid in urine. Absorption of m-xylene and excretion of m-methylhippuric acid were investigated under controlled conditions in ten volunteers aged 17-33 years. They were exposed to m-xylene vapours at concentrations of 100, 300, and 600 mg/m3. It was found that m-xylene vapour retained in the lungs tended to decrease at the end of the exposure. An exposure test was devised, based upon the results obtained during our investigations. The amount of xylene absorbed in the course of eight hours' work can be calculated with an accuracy of about +/- 8%.


Simultaneous determination by gas chromatography of the major metabolites in urine of toluene, xylenes and styrene. A gas chromatographic method has been developed to determine the following metabolites in urine simultaneously if necessary: hippuric acid from toluene or styrene; 3- and 4-methyl hippuric acids from xylenes; phenylglyoxylic acid and mandelic acid from styrene. Heptadecanoic acid is added to the urine as an internal standard and after ethyl acetate extraction from acidic solution, the trimethylsilyl (TMS) derivatives of the metabolites are formed and simultaneously analysed by gas chromatography on 3% OV-1 on 80/100 gas chrom Q (flame ionisation detector).


A simplified method for rapid determination of porphyrins by thin-layer chromatography. Free porphyrins, especially copro-, uro-, and protoporphyrin, were successfully separated by talc thin-layer chromatography (TLC), and were determined from the relationship of the weight and the area of porphyrins in the spot on the thin-layer plate. The results obtained from both the solvent extraction and the simple, rapid TLC method reported in this paper showed good correlation for coproporphyrin levels up to 400 microgram/1. The TLC method was applied to the determination of urinary coproporphyrin in lead workers and in rats.


A new method of carboxyhaemoglobin determination. A quick and accurate method of determining the concentration of carboxyhaemoglobin (COHb) in blood has been developed. The method uses a dual wavelength double beam spectrophotometer in the 1st derivative mode, linked to a digital voltmeter (DVM), with the two beams set 3 nm apart around an isobestic point of reduced haemoglobin (Hbred) and carboxyhaemoglobin at 579 nm. The 1st derivative mode measures the slope, and this slope is proportional to the concentration of COHb.


Determination of urinary manganese by the direct chelation-extraction method and flameless atomic absorption spectrophotometry. The direct chelation-extraction method, originally developed by Hessel (1968) for blood lead analysis, has been successfully applied to urinalysis for manganese. The analyses of 35 urine samples containing up to 100 microgram/1 of manganese from manganese-exposed workers showed that the data obtained by this method agree well with those by wet digestion-flame atomic absorption spectrophotometry and also by flameless atomic absorption spectrophotometry.


Arsenic exposure and mortality: a case-referent study from a Swedish copper smelter. An increased mortality from lung cancer, cardiovascular disease, haematolymphatic malignancy and cirrhosis of the liver has been reported among smelter workers and others exposed to arsenic. This study uses the case-referent (case-control) technique and is concerned with workers in a copper smelter in a complex work environment, characterised by the presence of trivalent arsenic in combination with sulphur dioxide and copper, and also with other agents. Lung cancer mortality was found to be increased about five-fold and cardiovascular disease about two-fold, showing a dose-response relationship to arsenic exposure. Mortality from malignant blood disease (leukaemia and myeloma) and cirrhosis of the liver was also slightly increased. This mortality pattern among the smelter workers is consistent with earlier reports. An increased mortality from cardiovascular disease in this type of industry is of particular interest as it has been reported only once before.


Characteristics of patients requesting reversal of sterilization. With the increased number of female patients seeking sterilization and the good chance of successful reversal using modern microsurgical techniques, it can be anticipated there will be a rise in the number of patients requesting reversal of sterilization. We have studied the characteristics of 36 patients who requested reversal of sterilization at the Royal Infirmary, Edinburgh, in the last five years. The majority of patients requested reversal on the basis of a new relationship. Other reasons included sexual problems and child death. Eighteen of the patients had clear evidence of marital disharmony at the time of sterilization. Seven of the patients had been admitted to hospital with a drug overdose since being sterilized.


A cervimeter for continuous measurement of cervical dilatation in labour-preliminary results. We briefly review instruments that have been used to measure cervical dilatation and describe the development of a new cervimeter. Curves derived from cervical dilatation and intrauterine pressure traces are presented in a form that clearly records the activity of the uterus and the response of the cervix to individual contractions. Our results suggest a new concept for behaviour of the cervix in labour.


Pregnancy in patients with bronchiectasis. Pulmonary function was monitored at regular intervals throughout pregnancy in three patients with bronchiectasis. They tolerated pregnancy well, and the underlying lung disease did not appear to have any adverse effect on the outcome of the pregnancy.


Copper intrauterine contraceptive devices in adolescent nulliparae. Copper intrauterine contraceptive devices (IUCDs) were used in 243 young nulligravidae attending a gynaecological clinic for schoolgirls in Uppsala between March 1973 and June 1975; 226 of these girls (93%) were seen again during December 1975 making a total of 3138 months of observation. At the time of follow up, 126 patients (55.8%) still had the IUCD in situ; 20% had expelled the IUCD and in another 24.2% the IUCD had been removed because of bleeding and/or pain or because of suspected pelvic inflammatory disease (7 patients). Twelve of the girls conceived during the period of observation making the Pearl index 4.6; nine of the conceptions occurred within nine months of the insertion of the IUCD. Unlike others we do not advocate the use of the IUCD for young nulligravidae.


Cyclic changes in the concentration of prostaglandin F2alpha in human uterine flushings. Prostaglandin F2alpha (PGF2alpha) was measured by radioimmunoassay in uterine flushings obtained from 41 women at various stages of the menstrual cycle. The concentration of PGF2alpha changed significantly during the cycle, with highest values in the mid secretory phase of the cycle (mean 9.1 ng/ml of uterine flushing, range 3.9 to 16.0 ng/ml, n = 9). The still unresolved role of intrauterine PGF2alpha is discussed in relation to luteolysis, myometrial contractility and implantation.


Oestradiol production by human graafian follicles cultured in vitro. Intact human Graafian follicles recovered during the preovulatory phase of the menstrual cycle and cultured in vitro, gave a mean oestradiol production of 33 ng/hour over periods of 4 to 92 hours. Treatment of follicles with human pituitary gonadotrophin or human chorionic gonadotrophin resulted in an increased production of oestradiol.