Conversion by rat brain preparation of lignoceric acid to ceramides and cerebrosides containing both alpha-hydroxy and nonhydroxy fatty acids. Effects of compounds which affect sphingolipid metabolism. Three synthetic compounds which affect sphingolipid metabolism in vitro were examined for their effects on the synthesis in a rat brain system of nonhydroxyceramide, hydroxyceramide, nonhydroxycerebroside, and hydroxycerebroside from [1-14C]ignoceric acid. These compounds are N-octanoyl-D-threo-p-nitrophenylaminopropanediol (Compound I), N-(2,3-epoxydecanoyl)-norephedrine (Compound II), and N-decyl-N'-glucosylthiourea (Compound III). In the presence of up to 0.5 mM Compound I, only the hydroxyceramide fromation increased, while the synthesis of the other three lipids decreased. Compound II strongly inhibited the formation of all four lipids at all concentrations tested (0.1 to 1 mM). Compound III increased the synthesis of both ceramides approximately 2-fold at 1 mM, but the conversion of lignoceric acid to both cerebrosides remained relatively unchanged. Several significant conclusions from these observations are discussed.


Solubilization and characterization of a platelet membrane ADP-binding protein. Previous studies have shown that platelet membranes bind radiolabeled ADP and have nucleoside diphosphokinase activity which transforms added ADP to ATP. In order to further characterize these reactions, the ADP-binding and nucleoside diphosphokinase activity of purified platelet membranes were solubilized by freeze-thaw injury followed by extraction with isotonic buffered saline. Up to 80% of membrane ADP-binding activity was solubilized along with 20% of the total membrane protein, a 4-fold purification. A Millipore filter binding assay was developed to detect the soluble binding protein using [3H]ADP as radioligand. Binding of [3H]ADP was rapid, reversible, saturable, and was destroyed by heat, trypsin digestion, and 1 mM N-ethylmaleimide. By Scatchard analysis, there was a single class of binding sites with a Kd of 3.8 x 10(-7) M. Unlabeled nucleotides competed with [3H]ADP with the following potency series: ATP = ADP greater than AMP greater than adenosine. The solubilized nucleoside diphosphokinase activity could be separated from ADP-binding activity by ultracentrifugation on 5 to 20% sucrose density gradients containing 0.6 M KCl suggesting that the activities reside on separate molecules. Hydrodynamic parameters were calculated for the binding protein by gel filtration and ultracentrifugation. The s20,w was 4.1, Stoke's radius 35 x 10(-8)cm, axial ratio (f/fo) 1.09, and the Mr = 61,000. The studies suggest that this platelet ADP-binding protein may act as the receptor for initiating ADP-induced aggregation and release.


Effect of trypsin on the cell surface proteins of hepatoma tissue culture cells. Characterization of a carbohydrate-rich glycopeptide released from a calcium binding membrane glycoprotein. Concentrations of trypsin that bring about aggregation of hepatoma tissue culture (HTC) cells also release from the cell surface an Mr = 55,000 glycopeptide fragment. This glycopeptide fragment also accumulates in the medium, including serum-free medium, as a normal consequence of membrane protein turnover. The trypsin-released glycopeptide is labeled when cells are grown in the presence of fucose or leucine before treatment of the cells with the protease. Similarly, the glycopeptide fragment can be labeled by reacting cells in situ by lactoperoxidase-catalyzed radioiodination or by tritiated borohydride reduction of cells treated first with neuraminidase and galactose oxidase. The tryptic glycopeptide fragment was purified by concanavalin A-Sepharose chromatography, and hydroxyapatite chromatography in the presence of dodecyl sulfate. The amino acid and carbohydrate composition was determined, as was the sensitivity of the purified glycopeptide to a variety of endo- and exoglycosidases. The purified glycopeptide contains an average of 17 sialic acid residues and hence, shows charge heterogeneity after electrophoresis in isoelectric focusing gels. The charge heterogeneity can be eliminated completely by treatment with neuraminidase. The glycopeptide after this treatment is homogeneous. The trypsin-sensitive membrane glycoprotein which is the source of the Mr = 55,000 glycopeptide was identified by two-dimensional gel electrophoretic analysis of labeled cells, treated or not treated with trypsin. This glycoprotein, which has an apparent molecular weight of 85,000 and forms a homodimer in the presence of calcium ions, was purified and its identity as the parent of the Mr = 55,000 glycopeptide was confirmed by showing that the same Mr = 55,000 fragment was released by trypsin from the purified glycoprotein as was released from the intact cells.


Binding of androgen receptor to prostatic chromatin requires intact linker DNA. Receptor-chromatin complexes were recovered from prostatic chromatin digested with micrococcal nuclease. The fragments of chromatin were separated on linear 7.6 to 76% (v/v) glycerol density gradients. With extensive digestion of DNA, receptor labeled with [1,2-3H]dihydrotestosterone was released from the chromatin. After 5% digestion of DNA to acid-soluble products, only a trace amount of labeled receptor was detected in the unbound form. In the latter instance, most of the labeled receptor was recovered from the gradients in association with five A260 peaks representing oligomeric and monomeric nucleosomes with a repeat length of 182 +/- 14 (mean +/- S.D.) base pairs. The concentration of receptors was highest in the A260 peaks, which contained large oligomers of nucleosomes, and lowest in fractions containing primarily monomer structures. Hence, the extent to which receptors remained bound to chromatin was dependent on the relative amount of intact, linker DNA present.


Metabolism of pyrazolo(3,4-d)pyrimidines in Leishmania braziliensis and Leishmania donovani. Allopurinol, oxipurinol, and 4-aminopyrazolo(3,4-d)pyrimidine. Leishmania donovani and Leishmania braziliensis grown in culture formed millimolar concentrations of allopurinol ribonucleoside 5'-monophosphate from [6-14C]allopurinol. In addition, allopurinol 1-ribonucleoside, oxipurinol riboside 5'-monophosphate, and three new metabolites of allopurinol, namely, 4-aminopyrazolo(3,4-d)pyrimidine ribonucleoside 5'-monophosphate and the corresponding di- and triphosphates (1-ribosyl 4-aminopyrazolo(3,4-d)pyrimidine 5'-diphosphate and 1-ribosyl 4-aminopyrazolo(3,4-d)pyrimidine 5'-triphosphate) were identified in the parasitic cells. They were formed via a unique amination reaction from 1-ribosyl allopurinol 5'-phosphate, analogous to the conversion of IMP to AMP. [6-14C]Allopurinol was incorporated into RNA of L. donovani in the form of 4-aminopyrazolo(3,4-d)pyrimidine. Adenine reversed the growth inhibition of allopurinol and prevented its metabolism to all of the ribonucleotide metabolites. L. donovani was 2- to 4-fold more active in its metabolism of allopurinol to ribonucleotides than L. braziliensis. 4-Aminopyrazolo(3,4-d)pyrimidine inhibited cell growth and resulted in high intracellular levels of 1-ribosyl allopurinol 5'-phosphate and smaller amounts of the 4-aminopyrazolo(3,4-d)pyrimidine ribonucleotides. The metabolism of allopurinol to 4-aminopyrazolo(3,4-d)pyrimidine ribonucleotides and its resultant cytotoxicity occurs in these parasitic protozoans, but not in mammalian cells.


Lipid requirements for the structure and function of the fatty acid synthetase complex from Ceratitis capitata. Lipid content of purified fatty acid synthetase preparations from the Dipterous Ceratitis capitata correlated with the enzyme activity. Delipidation of the enzyme by extracting with a series of organic solvents rendered a protein without any residual activity and the treatment with phospholipase A2 for 30 min reduced the activity to 10%. Addition of lipid classes to either the native enzyme or the phospholipase-treated preparation enhanced the activity in a different manner, phosphatidylethanolamine being the most effective lipid. The role of the lipids in the lipoprotein structure of the complex was studied by circular dichroism spectra of the native enzyme and in the presence of a concentration range of urea, guanidinium chloride, sodium dodecyl sulfate, sodium cholate, and sodium chloride. 3 M urea and 1.5 M guanidinium chloride induced a conformational transition of the lipoprotein that lost its alpha-helical structure at higher concentrations of both reagents. Sodium dodecyl sulfate and sodium cholate had little effect on the alpha-helical structure, although both reagents induced the loss of enzyme activity. Cholate had essentially the same effect as phospholipids on the maintenance of the native structure but it was unable to support the enzyme activity.


Dependence of the quantum efficiency for photolysis of carboxyhemoglobin on the degree of ligation. A combined stopped flow-laser photolysis apparatus was used to measure the quantum efficiency for removal of carbon monoxide bound to human hemoglobin as a function of fractional CO saturation. This flow-flash technique allows the properties of partially liganded hemoglobin molecules, which are sparsely populated under equilibrium conditions, to be conveniently studied. Experiments performed at pH 7 and 20 degrees C both in the presence and absence of phosphates gave a similar dependence of quantum efficiency on fractional saturation. The observed quantum efficiency was 0.90 +/- 0.06 at 10% saturation and decreased to 0.47 +/- 0.02 as full saturation was approached. An allosteric model in which Hb(CO)1 has a quantum efficiency of 0.99 while other liganded species have quantum efficiencies of 0.47 was used to produce a good simulation of the results.


Binding of human thrombin to cultured human endothelial cells. Binding of thrombin to monolayer cultures of human umbilical vein endothelium is studied. Binding is measured as inhibition by unlabeled ligand of the binding of 125I-thrombin to the cells. Radioactivity bound to cultures at equilibrium is measured after draining but not washing the cells. To correct for unremoved supernatant, 131I-albumin is included as a second label in the medium. Equilibrium between bound and free thrombin is attained within 1 min, and Scatchard analysis indicates a population of approximately 3 x 10(3) sites/cell with a dissociation constant of 10(-10) M, and a larger population with a dissociation constant greater than 10(-8) M. The two populations of sites are also indicated by a biphasic dissociation of bound label. Thrombin inactivated with diisopropyl fluorophosphate binds to the same receptor, with an affinity similar to that of active thrombin. Binding is unaffected by albumin (an acidic protein) and cytochrome c (a basic protein). Cultures of umbilical cord smooth muscle and fibroblasts bind thrombin at least 100 times more weakly than endothelium, and no binding to erythrocytes or a monolayer culture of mouse neuroblastoma is detected.


Interferon priming. Effects on interferon messenger RNA. The effects of priming mouse cells with interferon on the production of interferon and its mRNA were investigated. Interferon-treated (primed) mouse L929 cells produce 3 to 10 times more interferon than do nonprimed cells following induction with Newcastle disease virus. Interferon appears 2 to 4 h sooner in the primed cultures than in nonprimed cultures and interferon production by primed cells becomes resistant to inhibition by actinomycin D about 4 h sooner than interferon production in nonprimed cells. Interferon mRNA is detected in primed-induced cells about 2 h earlier than in nonprimed-induced cells. It reaches peak levels about 2 to 4 earlier in primed cells, but it also disappears sooner in primed cells. The total amounts of interferon mRNA isolated from primed-induced cells and nonprimed-induced cells were indistinguishable, by the methods utilized. Therefore, although primed cells can produce significantly more interferon and make interferon mRNA sooner than nonprimed cells, the total amount of interferon mRNA produced is apparently not increased, nor is its half-life prolonged in primed cells. Thus, enhanced interferon production in primed cells may result from enhanced efficiency of translation of interferon mRNA in the primed cells.


Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines. RNA polymerase II polypeptides present in [35S]methionine-labeled Chinese hamster ovary (CHO) cell extracts have been quantitatively immunoprecipitated with an anti-calf thymus RNA polymerase II serum. Analyses of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels indicated that the immunoprecipitated polymerase II of both wild type CHO cells and the alpha-amanitin-resistant mutant Ama1 had polypeptides of molecular weight 214,000, 140,000, 34,000, 25,000, 23,000, 20,500, and 16,500. In heterozygous alpha-amanitin-resistant/alpha-amanitin-sensitive hybrid CHO cells, growth in the presence of alpha-amanitin results in the inactivation of the alpha-amanitin-sensitive RNA polymerase II activity and a compensating increase in the activity of the alpha-amanitin-resistant enzyme. Determination of the rates of synthesis and degradation of RNA polymerase II polypeptides using [35S]methionine labeling and polymerase II immunoprecipitation demonstrated that this increase in activity of alpha-amanitin-resistant polymerase II resulted from a co-ordinate increase in the rate of synthesis of at least three polypeptides of RNA polymerase II. At the same time, there was an enhanced rate of degradation of the alpha-amanitin-inactivated RNA polymerase II polypeptides.


Characterization of a type IV procollagen synthesized by human amniotic fluid cells in culture. Fetal epithelioid cells, isolated from human amniotic fluid, synthesize and secrete a type IV-like procollagen characterized by a unique pattern of cyanogen bromide (CNBr)-produced peptides. The procollagen is disulfide-bonded and, after reduction, migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a doublet between collagen beta components and pro-alpha 1(I) chains. No conversion of the procollagen to collagen or to procollagen intermediates is observed in cell culture. The procollagen was purified by salt fractionation and ion exchange chromatography; its amino acid composition resembles that of collagenous proteins extracted from basement membranes, with a high 3- and 4-hydroxyproline and hydroxylysine content and low levels of alanine and arginine. The major products obtained after limited proteolytic digestion of the protein retain interchain disulfide bonds and, after reduction, migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis near intact pro-alpha 1(I) chains. The procollagen is secreted efficiently by amniotic fluid cells despite almost complete inhibition of peptidyl hydroxylation but, unlike type I procollagen, the secreted underhydroxylated chains lack interchain disulfide bonds. Since these cells also secrete fibronectin and elaborate an extensive extracellular matrix, the system should prove useful in the study of cell-matrix interactions.


Isolation and characterization of proteoglycans from porcine lungs. Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.


Refolding of the mixed disulfide of bovine trypsinogen and glutathione. The mixed disulfide of bovine trypsinogen and glutathione refolded with high yields at protein concentrations of 20 microgram/ml or less, at 4-25 degrees C, pH 8.0 to 8.7, in the presence of 3 to 6 mM cysteine under anaerobic conditions. The regenerated protein behaved as native trypsinogen as judged by gel exclusion chromatography, isoelectric focusing, and activation with bovine enterokinase or trypsin. However, refolded samples that were quenched with iodoacetate and analyzed by disc gel electrophoresis formed two components corresponding to trypsinogen and S-(carboxymethylcysteine)2-(179-203)-trypsinogen. The use of cysteine as a disulfide interchange catalyst caused reduction of the 179 to 203 disulfide bond, and quenching of the refolding mixture with iodoacetate produced the carboxymethylated derivative. The overall yield of the regenerated product was 70% and the half-time at 4 degrees C was 55 min.


The presence of an adrenodoxin-like ferredoxin and cytochrome P-450 in brain mitochondria. An iron-sulfur protein has been isolated from bovine brain mitochondria and purified 200-fold. The optical spectrum (peaks at 412 and 455 nm which disappear upon reduction) and the EPR spectrum (g values at 1.94 and 2.02) were typical for a ferredoxin. In reconstitution experiments, the protein could replace adrenodoxin in the cholesterol side chain cleavage reaction. The additional detection of cytochrome P-450 in brain mitochondria indicates that the isolated ferredoxin is part of a cytochrome P-450-dependent hydroxylation system.


Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit. The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.


Chiral [18O]phosphorothioates. The stereochemical course of thiophosphoryl group transfer catalyzed by nucleoside phosphotransferase. Nucleoside phosphotransferase from barley seedlings was used to catalyze the equilibration of adenosine-5'-[18O]phosphorothioate having the S configuration at phosphorus with [adenine-8-14C]adenosine to produce [adenine-8-14C]adenosine-5'-[18O]phosphorothioate and adenosine. The configuration of the chiral phosphorus in adenosine-5'-[18O]phosphorothioate which was used as the donor substrate was then compared with that of the [adenine-8-14C]adenosine-5'-[18O]phosphorothioate isolated from the reaction mixture. They were found to be the same, showing that the reaction proceeds with 99.7% retention of configuration of the [18O]phosphorothioate. This is interpreted to be indicative of the involvement of a thiophosphoryl-enzyme intermediate in the nucleoside phosphotransferase reaction. The synthesis of adenosine-5'-[18O]phosphorothioate having the R and S configurations at the phosphorus atoms is described.


Induction of cholera toxin receptors in cultured cells by butyric acid. Exposure of HeLa cells to sodium butyrate caused an increase in choleragen (cholera toxin) receptors as measured by increased binding of 125I-choleragen to the intact cells. The process was dependent on time and butyrate concentration; maximal increases (over 40-fold) were observed at 48 h and 5 mM sodium butyrate. Other short chain fatty acids were less effective in elevating choleragen receptors in the order: butyrate greater than pentanoate greater than hexanoate greater than propionate. Acetate and isobutyrate had no effect. The increase in toxin receptors caused by butyrate was reversible and occurred in serum-free medium. The affinity of choleragen for control and butyrate-treated HeLa cells appeared to be similar. Butyrate also induced an elevation in choleragen receptors in rat C6 glial and Friend erythroleukemic cells but not in a butyrate-resistant HeLa mutant. The increase observed in Friend cells paralleled the increase in ganglioside GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), the reported choleragen receptor. Although no GM1 could be detected in untreated Hela cells, small amounts were found in cells exposed to butyrate.


The degradation of proparathormone and parathormone by parathyroid and liver cathepsin B. Purified cathepsin B from porcine parathyroid glands was allowed to act upon radioactive bovine parathormone and proparathormone at various ratios of enzyme to substrate and for different times. The reaction products were isolated by ion exchange chromatography and analyzed by gel electrophoresis, amino acid composition, sequence analysis, and bioassay. The enzyme cleaved parathormone between residues 36 and 37 yielding a major carboxyl and amino fragment and appeared to cleave proparathormone at the same locus. The amino fragments were degraded further by removal of small peptides (possibly, di- or tripeptides) from their COOH termini. In contrast there was little if any degradation of the carboxyl fragment (residues 37 to 84). Despite the ease with which the enzyme cleaved the arginyl bond in the synthetic substrate benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide, it did not remove the near homologous NH2-terminal hexapeptide extension of proparathormone (Lys-Ser-Val-Lys-Lys-Arg-R)--a reaction that would lead to the formation of parathormone from proparathormone. Purified liver cathepsin B cleaved the hormonal substrates in a fashion identical with that of the parathyroid enzyme.


Purification to homogeneity of a beta-galactoside alpha2 leads to 3 sialyltransferase and partial purification of an alpha-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase from porcine submaxillary glands. Two different sialyltransferases (EC 2.4.99.1) have been resolved from Triton X-100 extracts of porcine submaxillary glands by affinity chromatography on CDP-hexanolamine agarose. The predominant sialyltransferase of this tissue, a CMP-N-acetylneuraminate: alpha-D-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase, has been obtained in a partially purified and stable form. A less abundant but highly active enzyme, a CMP-N-acetylneuraminate: beta-D-galactoside alpha2 leads to 3 sialyltransferase, was purified over 90,000-fold to homogeneity. Chromatography of the latter enzyme on Sephadex G-200 separated two noninterconverting forms, designated A and B, with Stokes radii of 51 A and 31 A, respectively. Both forms have equal specific activity toward lactose and contain a single polypeptide with a molecular weight of about 50,000 as estimated by gel electrophoresis. Form A appears to bind 1.18 g of Triton X-100 per g of protein, or nearly an entire detergent micelle per polypeptide, while Form B binds little or no detergent. The enzymatic properties of both forms are similar (Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) J. Biol. Chem. 254, 4444-4451) supporting the conclusion that Form A may represent the native sialyltransferase with an intact membrane-binding site, and Form B may be a large proteolytic fragment of Form A.


The calculation of some physical parameters of proteins from sucrose density gradient centrifugation data. A simplified procedure is described for the calculation of protein physical parameters from data obtained by sucrose density gradient centrifugation. The position of an uncharacterized protein relative to standard proteins is determined after centrifugation through linear 5-20% sucrose gradients prepared in both H2O and D2O. From these data, together with the density of each 5% sucrose buffer, the partial specific volume and standard sedimentation coefficient can be calculated without knowledge of rotor dimensions, angular velocity, time of centrifugation, or solvent viscosity. In the absence of specific solvent-induced changes in protein composition or conformation, the magnitude of error due to the approximations inherent in the method is estimated to be less than 2.3% in partial specific volume or less than 10-15% in sedimentation coefficient.


Enzymatic characterization of beta D-galactoside alpha2 leads to 3 sialyltransferase from porcine submaxillary gland. The substrate requirements, linkage specificity, and kinetic mechanism of a pure sialyltransferase from porcine submaxillary glands have been examined. The enzyme transfers sialic acid from the donor nucleotide, CMP-NeuAc, into the sequence NeuAcalpha2 leads to 3Galbeta1 leads to 3GalNAc, which is found in both glycoproteins and gangliosides. It forms only the alpha2 leads to 3 linkage with the disaccharide Gal/beta1 leads to 3GalNAc or antifreeze glycoprotein, which, along with asialoglycoproteins containing the sequence Gal/beta1 leads to 3GalNAcalpha1 leads to O-Thr/Ser, are the best acceptor substrates. Low molecular weight galactosides linked beta1 leads to 3 to glycose residues other than N-acetylgalactosamine are poor acceptors with relatively high Km values, while those in beta1 leads to 4 or beta1 leads to 6 linkages have both high Km and low Vmax. With glycoprotein and ganglioside acceptors this substrate specificity appears to be even more strict, with the sequence Gal/beta1 leads to 3GalNAc serving as the exclusive acceptor. Thus the present enzyme is not responsible either for the sequence, NeuAcalpha2 leads to 3Galbeta1 leads to 4GlcNAc, found in the asparagine-linked chains of certain glycoproteins, or for the synthesis of hematoside, NeuAcalpha2 leads to 3Galbeta1 leads to 4Glcbeta1 leads to 1Cer. Initial rate kinetic studies, with and without inhibitors, suggest that the transferase has an equilibrium random order mechanism.


Defective regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant. A somatic cell mutant of the CHO-K1 cell selected to be resistant to the killing effects of 25-hydroxycholesterol in the absence of cholesterol is shown to be defective in the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity by 25-hydroxycholesterol, cholesterol, and lipoproteins, thus maintaining the enzyme activity found in cells in the absence of exogenous sterol constitutively. The mutants phenotype is shown to be dominant with respect to the wild type. Actinomycin D and cycloheximide prevent the increase of HMG-CoA reductase activity that occurs in the CHO-K1 cell when cholesterol is removed from medium. Degradation of the enzyme, measured during inhibition of protein synthesis by cycloheximide, occurs at the same rate in the mutant as in the wild type. Kinetic studies indicate that the Km for two substrates, the activation energy, and a break in the Arrhenius plot are the same for HMG-CoA reductase determined in wild type and mutant cells. From these studies it is concluded that the mutant is defective in the regulation of synthesis of HMG-CoA reductase. Of the four processes which determine cellular cholesterol levels: biosynthesis, esterification, efflux, and uptake, only biosynthesis is altered, demonstrating that these processes are not co-ordinately controlled as has been suggested previously.


Purification and properties of heme oxygenase from rat liver microsomes. Heme oxygenase was purified to apparent homogeneity from liver microsomes of rats which had been treated with either cobaltous chloride or hemin to induce heme oxygenase in the liver and the purified preparations from either rats showed an apparent molecular weight of about 200,000 when estimated by gel filtration on a column of Sephadex G-200, and gave a minimum molecular weight of about 32,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hepatic heme oxygenase could bind heme to form a heme . heme oxygenase complex showing an absorption peak at 405 nm, and the extinction coefficient at 405 nm of the heme . heme oxygenase complex was 140 mM-1 cm-1. The heme bound to the hepatic heme oxygenase protein was easily converted to biliverdin when the complex was incubated with the NADPH-cytochrome c reductase system in air. The hepatic heme oxygenase appears to have characteristics essentially similar to those of the splenic heme oxygenase (Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224 and 4230). The heme oxygenase preparation which was purified from the cobalt-treated rats contained a small amount of cobaltic protoporphyrin, indicating that cobalt protoporphyrin was synthesized in these rats.


The initial stages of processing of protein-bound oligosaccharides in vitro. Following the rapid enzymatic transfer of an oligosaccharide (GlcNAc2Man9Glc3) from a lipid carrier to endogenous protein acceptors in membrane preparations from NIL fibroblasts, the transferred oligosaccharide chain undergoes processing. Protein-bound oligosaccharides, released from the polypeptide backbone by treatment with endo-beta-N-acetylglucosaminidase H, were analyzed by gel filtration and by susceptibility to alpha-mannosidase digestion. The initial stages of this processing in vitro consist of sequential excision of 3 glucose residues prior to the removal of mannose residues. The array of oligosaccharides generated in vitro by membrane preparations from NIL cells appears to be identical with processed oligosaccharides derived in vivo in intact NIL cells.


Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats. Mitochondrial and peroxisomal fatty acid oxidation were compared in whole liver homogenates. Oxidation of 0.2 mM palmitoyl-CoA or oleate by mitochondria increased rapidly with increasing molar substrate:albumin ratios and became saturated at ratios below 3, while peroxisomal oxidation increased more slowly and continued to rise to reach maximal activity in the absence of albumin. Under the latter condition mitochondrial oxidation was severely depressed. In homogenates from normal liver peroxisomal oxidation was lower than mitochondrial oxidation at all ratios tested except when albumin was absent. In contrast with mitochondrial oxidation, peroxisomal oxidation did not produce ketones, was cyanide-insensitive, was not dependent on carnitine, and was not inhibited by (+)-octanoylcarnitine, malonyl-CoA and 4-pentenoate. Mitochondrial oxidation was inhibited by CoASH concentrations that were optimal for peroxisomal oxidation. In the presence of albumin, peroxisomal oxidation was stimulated by Triton X-100 but unaffected by freeze-thawing; both treatments suppressed mitochondrial oxidation. Clofibrate treatment increased mitochondrial and peroxisomal oxidation 2- and 6- to 8-fold, respectively. Peroxisomal oxidation remained unchanged in starvation and diabetes. Fatty acid oxidation was severely depressed by cyanide and (+)-octanoylcarnitine in hepatocytes from normal rats. Hepatocytes from clofibrate-treated rats, which displayed a 3- to 4-fold increase in fatty acid oxidation, were less inhibited by (+)-octanoylcarnitine. Hydrogen peroxide production was severalfold higher in hepatocytes from treated animals oxidizing fatty acids than in control hepatocytes. Assuming that all H2O2 produced during fatty acid oxidation was due to peroxisomal oxidation, it was calculated that the contribution of the peroxisomes to fatty acid oxidation was less than 10% both in cells from control and clofibrate-treated animals.


Interactions of cartilage proteoglycans with hyaluronate. Inhibition of the interaction by modified oligomers of hyaluronate. Oligomers of hyaluronic acid were prepared by digestion of hyaluronic acid from rooster combs with testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35), leech head hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36), and with fungal hyaluronidase (hyaluronate lyase from Streptomyces hyalurolyticus). The oligomers were fractionated by gel permeation, using Sephadex G-50. Oligomers isolated after incubation of the hyaluronic acid with the testicular hyaluronidase were further modified. To prepare oligomers with N-acetylglucosamine at both ends, terminal nonreducing glucuronic acid residues were removed with beta-glucuronidase. Reducing terminal N-acetylglucosamine residues were removed by reaction under mildly alkaline conditions. The reducing terminal N-acetylglucosamine residues were also reduced with sodium borohydride to form N-acetylglucosaminitol. The potentials of the various oligosaccharides to bind to the proteoglycan from bovine nasal septum cartilage were estimated by determining their effectiveness as inhibitors of the proteoglycan-hyaluronate interaction. The present study shows that, to bind maximally to the proteoglycan, the hyaluronate oligosaccharide must be at least 10 sugar residues in length and be terminated at the nonreducing and reducing ends with a glucuronate residue and an N-acetylglucosamine residue, respectively. Sugar residues extended beyond this basic decasaccharide, do not interact with the hyaluronate binding site on the proteoglycan.


Mechanism of imipramine inhibition of platelet 5-hydroxytryptamine transport. Plasma membrane vesicles isolated from porcine blood platelets take up approximately 8 to 15 pmol of [3H]imipramine per mg of membrane protein. This apparent binding requires Na+ in the external medium and is reversed by 5-hydroxytryptamine and fluoxetine. The apparent KD for imipramine uptake is 23 nM, which agrees well with the KI for competitive inhibition of 5-hydroxytryptamine transport by imipramine. In contrast to 5-hydroxytryptamine transport, imipramine uptake is not dependent on transmembrane Na+ and K+ gradients and is insensitive to ionophores such as nigericin and gramicidin which dissipate these gradients. Although 5-hydroxytryptamine rapidly and competitively displaces imipramine from membrane vesicles, imipramine does not cause 5-hydroxytryptamine efflux and inhibits 5-hydroxytryptamine exchange. These results are consistent with the proposal that imipramine binds to the substrate site of the 5-hydroxytryptamine transporter but cannot be transported.


Synthetic glycolipids and the lectin-mediated aggregation of liposomes. Synthetic mannose-containing glycolipids utilizing the cholesterol nucleus as a lipid anchor, and either the 6-aminohexyl- or the 6-(6-aminohexanamido)hexyl-1-thio-alpha-D-mannopyranosides as the carbohydrate ligands, have been synthesized and incorporated into small unilamellar liposomes. Incorporation of these cholesterol-mannoside derivatives at concentrations up to 14 mol% apparently does not affect the physical characteristics of the liposomes. Addition of concanavalin A to a suspension of liposomes containing the long chain cholesterol-mannose derivative causes an increase in light-scattering at 360 nm. As the increase in absorbance is completely reversed by the addition of alpha-methylmannoside, aggregation rather than fusion of the liposomes appears to be occurring. Liposomes containing 14 mol % of the short chain (6-aminohexyl-) derivative are aggregated by concanavalin A indicating that the lectin can approach to within 10 A of the lipid bilayer. Preliminary results suggest that the aggregation of vesicles containing either the long or short chain derivatives is highly dependent on the density of the sugar in the membrane.


Migration of 40 S ribosomal subunits on messenger RNA when initiation is perturbed by lowering magnesium or adding drugs. Migration of 40 S ribosomal subunits on messenger RNA, detected previously in experiments using the antibiotic edeine (Kozak, M., and Shatkin, A.J. (1978) J. Biol. Chem. 253, 6568-6577) has now been observed in the presence of other inhibitors of initiation. 40 S subunit migration has been detected in both wheat germ and reticulocyte lysates treated with edeine, pactamycin, or sodium fluoride. The variety of structurally unrelated inhibitors that mediate this effect argues against the interpretation that migration is a drug-induced artifact. Indeed, limited migration of 40 S ribosomes occurs upon simply lowering the magnesium concentration, in the absence of inhibitors. Thus, migration seems to be an inherent property of 40 S ribosomal subunits and might be involved in the mechanism by which eukaryotic ribosomes select initiation sites in messenger RNA.


Stereochemical specificity of the biosynthesis of the alkyl ether bond in alkyl ether lipids. The stereochemical course of the formation of the alkyl ether bond in alkyl ether lipids was investigated through the synthesis of stereospecifically labeled acyl R- or S-[1-3H]dihydroxyacetone 3-phosphate (DHAP) starting from L-glyceraldehyde. It was demonstrated directly that the formation of the alkyl ether bond results in the stereospecific exchange of the pro-R C-1 hydrogen of DHAP with a proton of water. The configuration of the hydrogen that is retained on C-1 after formation of the alkyl ether bond was also investigated. The alkyl ether lipid was degraded, and the DHAP backbone isolated as glycerol, converted to DHAP via glycerol 3-phosphate and treated with either aldolase or triose phosphate isomerase. The results demonstrated that the retained hydrogen on C-1, which was pro-S in the starting substrate, was pro-S in the product alkyl ether.


Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors. The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.


The complete primary structure of a proline-rich phosphoprotein from human saliva. The complete amino acid sequence of a calcium-binding "proline-rich phosphoprotein," named Protein A, from human saliva was determined by automated and manual Edman degradation of peptides obtained by enzymatic and chemical cleavage of the intact protein. The NH2-terminal pyrrolidone carboxylic acid was identified by means of NMR. The protein consists of 106 amino acids, including 24 residues of proline. The NH2-terminal 32 residues contain 13 of the 15 negatively charged residues including 2 phosphoserines, but only 1 proline. In spite of a high concentration of proline in the COOH-terminal part of the molecule, the longest oligoproline sequence is tetraproline. The protein contains a number of repeated sequences and there are also several sequences of 3 or 4 residues identical with known sequences of collagen, but the characteristic occurrence of glycine in every third position in collagen is not found in salivary Protein A.


Properties of a soluble polyprenyl phosphate: UDP-D-glucose glucosyltransferase. A soluble enzyme that catalyzes the transfer of D-glucose from UDP-D-glucose to dolichyl phosphate has been prepared by sonic oscillation of Acanthamoeba castellani cysts. The product of catalysis is dolichyl beta-D-glucosyl phosphate. The enzyme requires a divalent cation, either magnesium or manganese, and the presence of a reducing agent for maximum activity. Solanesyl phosphate and ficaprenyl phosphate are alternative substrates, apparently at lower rates, but GDP-D-glucose, UDP-D-glucuronic acid, UDP-N-acetyl-D-glucosamine, and UDP-D-xylose are not substrates. The temperature optimum is 30 degrees C, the pH optimum is pH 7.0, the Km for UDP-Glc is 9.1 microM and for dolichyl phosphate it is 4.5 microM. Uridine monophosphate and UDP are inhibitors of the reaction, UDP causing reversal and UMP being a competitive inhibitor of UDP-Glc with a Ki of 62 microM. The enzyme can be stored indefinitely below -20 degrees C, is stable for several days at 4 degrees C, but is half-inactivated within 2 h at 30 degrees C and completely inactivated within 10 min at 52 degrees C.


Urinary glycopeptides of fucosidosis.

Urinary glycopeptides of fucosidosis. Fucosidosis is unique among congenital exoglycosidase deficiencies, because not only oligosaccharides but large amount of glycopeptides are excreted in the urine. The structures of 22 major glycopeptides isolated from urine of a fucosidosis patient were determined. One of the prominent features common to all these glycopeptides was that they all contain 1 fucosyl residue at either C-3 or C-6 position of the N-acetylglucosamine which is linked to asparagine. Possibly, accumulation of glycopeptides in fucosidosis urine is caused by the inability of human endo-beta-N-acetylglucosaminidase to cleave the asparagine-linked sugar chains, which have a fucose at the innermost N-acetylglucosamine residue.


Cleavage of a COOH-terminal hydrophobic region from D-alanine carboxypeptidase, a penicillin-sensitive bacterial membrane enzyme. Characterization of active, water-soluble fragments. D-Alanine carboxypeptidase (CPase), a detergent-soluble penicillin-sensitive membrane enzyme of Bacillus stearothermophilus, Mr = 46,500, was digested with either trypsin or alpha-chymotrypsin to yield water-soluble fragments, designated T-CPase and Chy-CPase, respectively, each of Mr = approximately 45,000. These fragments were generated and purified in milligram quantities by digestion of CPase covalently immobilized on a penicillin affinity column. They retained full enzymatic activity, became significantly more resistant to thermal inactivation, and lost micellar detergent binding upon proteolysis. Each was derived from CPase by loss of a COOH-terminal hydrophobic peptide. CPase was reconstituted into bacterial lipid vesicles in an enzymatically active form. Penicillin-binding sites were equally distributed on both sides of the lipid bilayer, suggesting a random orientation of the CPase molecules. Neither T-CPase nor Chy-CPase reconstituted into lipid vesicles when treated in an identical manner. CPase was slowly cleaved from the surface of these vesicles by either trypsin or alpha-chymotrypsin, yielding T-CPase and Chy-CPase, respectively. These results demonstrate that CPase is comprised of a water-soluble catalytic domain and a COOH-terminal hydrophobic region which mediates the anchoring of this enzyme to the bacterial membrane.


Preliminary crystallographic data for Bowman-Birk inhibitor from soybean seeds. A well characterized soybean protease inhibitor, the Bowman-Birk inhibitor, has been crystallized at room temperature in the presence of polyethylene glycol 4000 by vapor diffusion against an ammonium sulfate solution containing 2-methyl-2,4-pentanediol. An x-ray diffraction study reveals that the inhibitor crystallizes in a hexagonal unit cell of symmetry P6122 (or P6522) and dimensions a = b = 91.36(2) A and c = 63.92(2) A. Each of the 12 asymmetric units contains 2 molecules of molecular weight 8000. The crystal, which diffracts barely to 3-A spacings, is fairly stable to x-irradiation and has a solvent content of approximately 52% by volume.


Free minicircles of kinetoplast DNA in Crithidia fasciculata. The major form of kinetoplast DNA in Crithidia fasciculata is a network which contains thousands of minicircles linked together in a two-dimensional array. This paper reports the existence of free minicircles in Crithidia which by several criteria are identical to those in networks. They are the same size (about 2500 base pairs), and they yield the same products upon digestion with restriction enzymes. About 0.4% of the minicircles in exponentially growing nonsynchronized cells are free and the remainder are in networks. After a 5-min pulse with [3H]thymidine, above 10% of all of the incorporated radioactivity in the cell is in free minicircles, and the minicircles have a higher specific radioactivity than the average of other DNAs in the cell. Three-branched structures, which resemble Cairns-type replication intermediates, are occasionally observed by electron microscopy. Kinetic studies of the incorporation of [3H]thymidine into free minicircles indicate that they turn over, and this turnover was confirmed by a pulse-chase experiment. These properties of free minicircles suggest that they may be intermediates in the replication of network minicircles.


Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data. Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.


Heparinized styrene-butadiene-styrene elastomers. A heparinized high-strength elastomer has been developed which is potentially useful as a nonthrombogenic vascular prosthesis. A surface hydroxylated styrene-butadiene-styrene (SBS) block copolymer with at least 40% extent of reaction after glow-discharge cleaning was coated with a 20% acetylated polyvinyl alcohol/heparin mixture containing glutaraldehyde and magnesium chloride. After curing at 80 degrees C for 100 min, the polyvinyl alcohol, heparin, and hydroxylated SBS were covalently bound to each other by acetal bridges. The effects of the various substrate and coating parameters were optimized to achieve very strong adhesion between the coating layer and the surface hydroxylated SBS. Heparin was not leached from the surface of the new material using 3M saline at pH 7.4 despite a detection limit of 10(-5) micrograms heparin/cm2 min. Prolonged partial thromboplastin times of greater than 1200 sec were observed (control: PTT = 120 sec). Preliminary ex vivo testing using a simple arteriovenous shunt in the leg of a rabbit showed good thromboresistance. The heparinized SBS shunt chamber remained patent for more than two hours without desorption of heparin. It was concluded that surface hydroxylated SBS heparinized by acetal coupling owed its thromboresistance to the heparin covalently bound to the surface and not to a microenvironment of heparin in solution at the blood/material interface.


Effect of an in vivo environment on the strength of bone cement. Bar-shaped polymethylmethacrylate test specimens removed from rabbits after implantation for times up to 26 months showed a significant change in fracture stress as determined by three-point bending in the period between 12 and 26 months. There were no adverse findings in the tissue which developed around the bone-cement test bars.


Time-dependent phase changes in Cu-rich amalgams. Cu-rich amalgams represent a significant change in amalgam alloy formulations which may result in enhanced performance. Significantly, these systems reduce or eliminate the gamma2 phase. This work studied the phase changes which occurred from one hour to six months in 13 commercial amalgams by x-ray diffraction. This included 9 two-particle or blend systems and 4 single-particle systems. Gamma2 was found in all systems, but was reduced or eliminated on storage at 37 degrees C at rates which varied from system to system. During gamma2 elimination, the Cu6Sn5 content correspondingly increased, indicating that solid-state reaction occurs for up to six months in some systems.


13C NMR spectroscopic analysis of poly(electrolyte) cement liquids. 13C NMR spectroscopy has been applied to the analysis of carboxylic poly-acid cement liquids. Monomer incorporation, composition ratio, sequence statistics, and stereochemical configuration have been considered theoretically, and determined experimentally, from the spectra. Conventionally polymerized poly(acrylic acid) has an approximately random configuration, but other varieties may be synthesized. Two commercial glass-ionomer cement liquids both contain tartaric acid as a chelating additive but the composition of their poly-acids are different. Itaconic acid units, distributed randomly, constitute 21% of the repeating units in one of these polyelectrolytes.


The influence of grain size on the fatigue behavior of annealed 316 LVM stainless steel. Experimental tests demonstrate that there is a sharp drop in resistance to fatigue fracture when the grain size is increased from 75 to 150 micrometers. This is at least a partial explanation for fatigue fractures of the stems of total hip-joint prostheses reported in the literature. It is also shown that plastic strains associated with fatigue stressing increase with increasing grain size so that loosening of the bone cement embedment may also be part of the premature failure process.


Bending properties of wire-reinforced bone cement for applications in spinal fixation. PMMA beam specimens were tested in four-point bending to determine if the bending strength of acrylic bone cement, as used in posterior spinal fusion, could be improved by metal-wire reinforcement. The result showed that the load-carrying capacities of 1- and 0.5-mm diam stainless-steel-wire-reinforced PMMA specimens in bending were significantly higher than similar unreinforced normal PMMA samples. On an average, steel reinforcement comprising approximately 1% of the cross-sectional area of the PMMA specimens caused a 15% increase in bending strength. Even after the cement fractured, the reinforcing wires still sustained an appreciable amount of bending moment, thus preventing catastrophic failure of cement alone.


Role of catheter surface morphology on intravascular thrombosis of plastic catheters. The role of catheter surface roughness in initiation and propagation of intravascular catheter thrombosis was studied in dogs, utilizing the scanning electron microscope. Catheters were inserted in major arteries and veins for 200 min and the animals then sacrificed. Catheter segments, both with and without associated thrombi, were scrutinized with the scanning electron microscope. No correlation was found between catheter surface roughness and thrombus formation, suggesting that the inherent chemical characteristics of the catheter is most important in the initiation of intravascular catheter thrombus formation.


The influence of final mercury content on the characteristics of a high-copper amalgam. In this study, specimens of a high-copper amalgam, prepared at different final Hg contents, were examined in several different experiments. The results showed that as the Hg content is increased, a point is reached beyond which creep and the amount of Sn in gamma1 exhibit a sudden increase. As the Hg content is increased further, gamma2 can be detected. These phenomena can be explained by the hypothesis that insufficient Cu is present at higher Hg contents to combine with Sn to form Cu6Sn5.


Sustained drug delivery systems. I. The permeability of poly(epsilon-caprolactone), poly(DL-lactic acid), and their copolymers. The maximum steady state flux, diffusion coefficients, and solubilities of five contraceptive steroids in homopolymers and copolymers of epsilon-caprolactone and DL-lactic acid were determined. The permeabilities of polymers of epsilon-caprolactone were comparable to silicone rubber and, by inference, are suitable for the construction of drug delivery devices. Poly(DL-lactic acid) was 10(4) times less permeable, although its permeability was significantly enhanced by additives.


Management of severe spondylolisthesis in children and adolescents. Forty-three patients with a fifth lumbar-first sacral spondylolisthesis of 50 per cent or greater were reviewed. Four had been treated non-operatively; eleven, by arthrodesis; eighteen, by decompression and arthrodesis; and ten, by reduction and arthrodesis. The angle of slipping (measurement of the kyphotic relationship of the fifth lumbar to the first sacral vertebra) was found to be as important a measurement as the percentage of slipping in measuring instability and progression of slipping. Hamstring tightness did not correlate with neural deficit. Arthrodesis alone, even in the presence of minor neural deficits, tight hamstrings, or both, gave relief of pain and resolution of neural deficits and tight hamstrings. Our experience with a limited number of patients suggests that management by postoperative extension casts may achieve a significant reduction in percentage of slipping and in angle of slipping. Progression of the spondylolisthesis may occur following a solid arthrodesis.


Surgical management of thoracic kyphosis in adolescents. Twenty-seven patients of a group of twenty-nine who underwent posterior spine fusion using Harrington compression instrumentation for Scheuermann's disease or postural roundback deformity were reviewed with a mean follow-up of 27.6 months. The mean curve before operation was 72 degrees and at review, 46.1 degrees. The mean loss of correction in the fusion from initial examination to final follow-up was 5.7 degrees. This procedure can achieve satisfactory correction of deformity as well as pain relief. We think that Harrington compression instrumentation and spine fusion is indicated in selected cases of roundback deformity.


Ipsilateral concomitant fractures of the hip and femoral shaft. Fifty-two cases of concomitant ipsilateral fractures of the hip and femoral shaft were reviewed from the literature and twenty-one of our own were added. Thirty-one per cent of the femoral-neck fractures reported in the literature and one of our own were missed on initial evaluation. Examples of non-union and aseptic necrosis were all from the previously reported cases and occurred in the patients in whom there was a delay in diagnosis and treatment of the fracture of the neck. Nine of our patients had associated ipsilateral knee injuries. In nine pulmonary complications developed, all while in skeletal traction. If the patient survived the initial injury, the prognosis was surprisingly good. We concluded that intracapsular hip fractures should be treated by internal fixation. Good long-term results were the rule in the diaphyseal fractures. Internal fixation of both fractures should be considered in patients with severe multisystem trauma, in the very elderly, and in those with ipsilateral knee injuries. Ender pins were useful and were employed in five of our patients.


Intra-articular acetabular labrum: a possible etiological factor in certain cases of osteoarthritis of the hip. In eight patients with so-called idiopathic degenerative arthritis of the hip the acetabular labrum, at surgery, was found to lie in the articulation between the femoral head and the acetabulum. In none of the patients was there a history or roentgenographic evidence of congenital dysplasia or congenital dislocation. None of the patients had had a closed reduction or manipulation of the hip during infancy, childhood, or adolescence. No patient had had a traumatic dislocation. The findings suggest that the intra-articular labrum was a developmental abnormality and we postulate that this abnormality was the cause of the degenerative arthritis.


Lengthening of the elbow flexors in cerebral palsy. A procedure has been devised for treatment of the inability to extend the elbow due to either a fixed contracture of the flexor muscles or increased involuntary flexor-muscle tone and spasticity. The operation consists of lengthening all of the primary elbow-flexor muscles. It has been used successfully in thirty-two elbows of twenty-six children with cerebral palsy who were followed for an average of four years (range, two through six years). The average gain in extension was 40 degrees, and there was no loss of the patient's ability to flex the elbow or supinate the forearm. No vascular or neural complications were encountered. Independence in feeding was achieved in four patients. Seven patient acquired the ability to engage in sports and six, who previously had been unable to walk independently due to inability to handle crutches, could do so following this procedure.


Gross and histological abnormalities of the talus in congenital club foot. Gross and histological abnormalities were demonstrated in a club-foot talus from a boy with multiple congenital anomalies who died when he was nine days old. Both tali were studied, the one from the club foot and the one from the normal foot. The gross anomalies involved the smaller size of the club-foot talus and the increased medial deviation of a stunted, misshapen head and neck region. Serial histological sections of both tali allowed for a three-dimensional geometric appreciation of both bones and an assessment of the nature and extent of histological and cytological features. The ossification center of the club-foot talus was absolutely and relatively smaller than that of the normal talus. It was eccentrically positioned, being more lateral and anterior than that of the normal talus. The marked histological abnormalities seen in the head and neck region of the club-foot talus involved extensive breaching of the endochondral sequence by vessels. The posterior aspect of the endochondral sequence and ossification center was normal. The extra-osseous and intra-osseous blood supply of the two tali was normal with the exception of the increased and irregular breaching of the endochondral sequence in the club-foot talus. This study demonstrates histological abnormalities in the head and neck region of the club-foot talus, which was most abnormal grossly. The eccentric position of the secondary ossification center as well as its related vascular abnormalities do not support a theory of developmental arrest of the talus but appear sufficiently abnormal to support the theory of a primary defect in the cartilage anlage. Clinically, one must bear in mind that early open reduction of the talocalcaneal navicular joint in a foot such as this would have served to reposition the navicular onto a talus that still was structurally abnormal.


The effect of hypotensive anesthesia on cephalothin concentrations in bone and muscle of patients undergoing total hip replacement. Serial blood, muscle, and bone concentrations of cephalothin following intravenous infusion were determined in forty-eight patients during total hip replacement. Thirty patients received hypotensive anesthesia and the remaining eighteen received anesthesia without induced hypotension. The concentrations of cephalothin were determined during the first (early) and second (late) hours of the operation. Late serum levels were lower in the patients who had induced hypotension. In all patients at least one bone specimen contained measurable amounts of cephalothin. When pentolinium (Ansolysen) was used to induce hypotension, the late serum and early bone cephalothin concentrations were lower, while use of trimethaphan (Arfonad) did not appear to result in lower cephalothin concentrations in serum, muscle, or bone compared with patients with normotensive anesthesia.


Functional adaptation of bone to increased stress. An experimental study. In young pigs walking normally, we recorded the principal compressive strains (epsilon 2) as revealed by strain gauges attached to the shaft of the radius. We found no correlation between the mean principal compressive strain and either age or weight in normal animals over the three-month time span of the experiment. Directly after unilateral removal of the ulnar diaphysis, the principal compressive strain on the radial shaft increased by two to two and one-half times its normal value. The increased level of strain in the radius caused by ulnar ostectomy resulted in a rapid and substantial remodeling response in the radius, but three months after ulnar ostectomy, the area of bone enclosed by the periosteal perimeter of the overstrained radius approached the value for the radius and ulna together in the contralateral limb, as did the cross-sectional area of the bone tissue in the mid-shaft of that radius. Three months after unilateral ostectomy, the principal compressive strain was not significantly different in the left and right radii and approached normal values.


Movement patterns of interosseus-minus fingers. The patterns of isolated metacarpophalangeal-joint flexion and isolated interphalangeal-joint extension movements in eighty fingers with intact extrinsic and lumbrical muscles but no interossei (so-called interosseus-minus fingers) were studied. The pattern of metacarpophalangeal flexion resembled that seen in fingers with paralysis of all intrinsic muscles. From this it was inferred that in the interosseus-minus finger the lumbrical muscles are inactive during this movement. The pattern of isolated interphalangeal extension in the interosseus-minus fingers showed an improvement over that in totally intrinsic-minus fingers, suggesting that the lumbrical muscles reduce the extending forces at the metacarpophalangeal joint to some extent. Active participation of the interosseus muscles appears to be necessary for carrying out the movements of isolated metacarpophalangeal flexion and interphalangeal extension.


Recurrent anterior dislocation of the shoulder. Results after the Bankart and Putti-Platt operations. A postoperative follow-up of one and one-half to ten years was obtained in 114 of 121 patients who underwent either a Bankart or a modified Putti-Platt procedure. In the forty-six patients who had a Bankart operation, there was one recurrence (2 per cent) compared with thirteen recurrences (19 per cent) in the sixty-eight patients who had a modified Putti-Platt procedure. Twelve (36 per cent) of the latter recurrences occurred in patients who were twenty-five years old or younger at the time of the operation and only one occurred in the patients who were more than twenty-five years old. The restriction of outward rotation after shortening of the subscapular tendon averaged 6 degrees as compared with 16 degrees after a Bankart operation. A modified Putti-Platt procedure (shortening of the subscapular tendon) is recommended only for patients who are more than thirty years old. It does offer certain advantages for them because of the simplicity of the procedure and the short duration of the operation.


The semiconstrained total shoulder arthroplasty. In a two-year follow-up on sixteen total shoulder arthroplasties done with the semiconstrained Stanmore prosthesis, we  found that patients with rheumatoid arthritis or osteoarthritis of the glenohumeral joint noted a greater relief of pain, were more satisfied with their surgery, used fewer analgesics, and had a greater increase in range of motion than patients with traumatic arthritis. Patients with irreparable rotator-cuff injury demonstrated the poorest range of motion postoperatively. Younger patients gained significantly more motion than older patients. All patients continued to have difficulty with lifting heavy loads. Complications consisted of one patient with a chronically dislocating prosthesis, one patient with a deep wound infection, and one with a postoperative wound dehiscence. The average follow-up was 31.4 months, ranging from twenty-four to fifty-three months.


Ligament injuries in the knees of children. Injuries of the medial collateral ligament in children are rarely reported. We are describing six tears of the medial collateral ligament in which an operative repair was done. The patients' ages were six to eleven years. Five were boys and one, a girl. The follow-up (seven months to thirteen years) showed normal results in three; good, in two; and fair, in one. Three patients also had had an injury to the anterior cruciate ligament or an avulsion injury to the anterior tibial spine.


Congenital deficiency of the fibula.

Congenital deficiency of the fibula. Ninety-seven limbs, in eighty-one patients, with a diagnosis of congenital deficiency of the fibula have been reviewed. A classification was devised to distinguish the minimal hypoplasia of the fibula (Type I) from the well-known complete absence (Type II). Congenital anomalies of the femur were present in 76 per cent of patients with Type I deficiency and in 59 per cent with Type II. The shortening of the limb was by 13 per cent in Type I and by 19 per cent in Type II, and the percentage shortening was fairly constant during growth. A detailed description of the spectrum of other congenital anomalies was found to be characteristic: for example, the ball and socket formation of the ankle, tarsal coalition and anomalies of the foot. The treatment aimed simply to equalise leg length in Type I deficiency, while amputation of the foot and the fitting of a prosthesis were necessary in Type II to obtain satisfactory function.


A histological study of idiopathic avascular necrosis of the head of the femur. We have studied the histological appearances of forty femoral heads with idiopathic avascular necrosis. The characteristic histopathological changes of recurrent necrosis were present in 83 per cent. Recurrent necrosis occurred widely after revascularisation had progressed as far as the subchondral zone. The aetiology of idiopathic avascular necrosis of the femoral head may be a chronic condition which produces repeated infarction. The deformation and incomplete revascularisation of the femoral head may be due to repeated episodes of infarction as well as to mechanical factors related to weight-bearing.


Low-friction arthroplasty for healed septic and tuberculous arthritis. Forty hips, which had previously been the site of tuberculous or pyogenic arthritis and which had later developed a degenerative arthritis, were treated by low-friction arthroplasty some forty years after the original infection. The results suggest that, when healing of the primary infection has been followed by a long period of quiescence with acceptable function before the onset of degenerative change, the arthroplasty can be confidently expected to result in greatly improved function and that this improvement is long-lasting.


The treatment of femoral fractures by cast-brace and early walking: a review of seventy-nine patients. Seventy-nine cases of fracture of the femoral shaft treated by cast-brace and early walking have been reviewed. Discrepancy in femoral length was assessed by scanogram. The cases were analysed to relate the incidence of shortening greater than 2 centimetres to the type and site of the fracture, and the time which elapsed from injury until the cast-brace was applied. Such shortening was encountered most frequently when the cast-brace was applied within the first two weeks from injury or after six weeks and in those patients with comminuted fractures of the middle third of the femoral shaft.


Arthrodesis of the shoulder in rheumatoid arthritis: a review of forty-one cases. Forty-one arthrodeses of the shoulder in thirty-nine patients suffering from rheumatoid arthritis (thirty women, nine men) have been reviewed. Using internal fixation and external splints the position of the shoulder was maintained in 55 degrees of abduction, 25 degrees of horizontal flexion and enough internal rotation to allow the patient to reach the mouth. The mean period of immobilisation in a thoracobrachial splint was nine weeks, and 90 per cent of the shoulders had solid bony fusion at review. After arthrodesis the total range of scapulothoracic movement improved by about 60 per cent, giving results rated as excellent in fifteen cases (36 per cent), as good in thirteen (32 per cent) and as fair in thirteen (32 per cent). Arthrodesis can be recommended as an easy, cheap and reliable method of treating a shoulder which has been severely destroyed by rheumatoid arthritis.


The results of operation for ulnar neuritis. The records of 110 cases of ulnar neuritis in 100 patients have been reviewed an average of 4.4 years after anterior transposition, or release of the aponeurosis. Seventy of the patients were reviewed personally. In over half the cases no precipitating cause was apparent. At operation the nerve was constricted by the flexor carpi ulnaris aponeurosis in fifty cases but in twenty-five no abnormal pathology was found. Recovery was greatest when operation was performed within three months of the onset of symptoms. In those cases where no abnormality was found, and those in which adhesions in the postcondylar groove involved the nerve, simple release was less effective than anterior transposition. It is therefore recommended that release be restricted to patients with a short history and with an obvious aponeurotic constriction unaccompanied by adhesions. Anterior transposition is the operation of choice where no abnormality is seen or where the nerve is dislocated, compressed or tethered proximal to the aponeurosis of flexor carpi ulnaris.


Sleeve fractures of the patella in children: a report of three cases. The clinical characteristics of sleeve fractures of the patella are described and the methods of treatment are reviewed. Attention is drawn to the fact that the diagnosis may be missed because the distal bony fragment may be so small as not to be detectable by radiography. This is important because a large fragment of articular cartilage also separates. The best result was obtained from reconstitution of the extensor apparatus by internal fixation of the patellar fracture with repair of the quadriceps expansion.


An electromyographic investigation of subluxation of the patella. An electromyographic investigation of patients with subluxation of the patella has been carried out on the parts of the extensor apparatus which actively contribute to the alignment of the patella, both before and after the operation to correct this disorder. The electromyographic pictures have revealed a sharp fall in the activity of the vastus medialis, with full recovery to normal values after a corrective operation. Even if the aligning function of the patella is altered by a variety of factors, the present study confirms the importance of the vastus medialis in the pathogenesis of malalignment of the extensor mechanism.


The "small patella" syndrome.

The "small patella" syndrome. Twelve closely related members of one family were found to have small or absent patellae. Seven of these patients also had abnormalities of the pelvic girdle and upper femora. There was a resemblance to the nail--patella syndrome in the patellar abnormalities but not in the associated conditions, and it would therefore appear that the syndrome described is a new variety of congenital abnormality of the knee.


Hallux valgus in the younger patient: the structural abnormality. Standardised radiographs of the weight-bearing foot were analysed in fifty young patients undergoing osteotomy of the first metatarsal for hallux valgus. True metatarsus primus varus was not found more frequently than in a control series. The intermetatarsal angle was significantly greater in affected feet compared with controls. The structural abnormality in hallux valgus in the young is therefore due to a valgus disposition of the second and subsequent metatarsals, rather than varus inclination of the first metatarsal.


Old fibrin coagula and their ossification in simple bone cysts. Old calcified fibrin coagula are frequently found in simple bone cysts. They provide a scaffold on which new bone is laid down, in a process analogous to endochondral ossification. It is suggested that these coagula are derived in substantial part from the plasma-like contents of the cyst, after the release of plasma-clotting factors as the result of injury. Major haemorrhage is not involved and in many cases there is no antecedent fracture. The phenomenon is not seen in other common cystic conditions of bone and its recognition is thus helpful in the histological diagnosis of simple bone cyst. Cystic bone infarcts and their possible confusion with simple bone cysts are also briefly discussed.


The effects of methylprednisolone acetate in the treatment of bone cysts. Results of three years follow-up. The treatment of bone cysts by topical injection of methylprednisolone acetate was initiated at the end of 1973, and the late results are reported in this paper. In seventy-two cases followed up for one to three years favourable results have been obtained in about 90 per cent. The technique of local injection and the surgical equipment employed, in the case of focal recurrences, are considered. With this method, surgical treatment of bone cysts in youth is seldom necessary.


School screening for scoliosis in Athens. A randomly selected sample of 3494 children evenly representing a total population of 37 391 schoolchildren aged between eleven and twelve was screened for idiopathic scoliosis by the bending test. Ten per cent of the children showed clinical evidence of scoliosis and 6.4 per cent had positive radiological findings. Small curves were equally distributed between the two sexes, while large ones were more common in girls. Children with fair hair and blue eyes showed a higher percentage of positive findings than children with a dark complexion. A group of 112 children with curves between 7 and 16 degrees were followed up for an average of nineteen months. The pattern and evolution of these curves showed their close relationship to established scoliosis. Potential factors of instability and deterioration of the curves were evaluated.


The role of the periosteum in the growth of long bones. An experimental study in the rabbit. Hemicircumferential division of the periosteum was performed on the upper tibia of the rabbit. Division of the medial side regularly caused a valgus angulation, but other injuries about the upper tibia had no effect. The cause of deformity after periosteal damage is discussed.


Autoradiography of technetium-labelled diphosphonate in rat bone. The passage of technetium-labelled methylene diphosphonate (99mTc-MDP) across rat bone was examined by autoradiography. The autoradiographs showed that shortly after an injection of the bone-seeking agent there was activity outside the bone, within the bone marrow and also adjacent to the highly vascular epiphysial plate; the distribution of the isotope in the incubated bone appeared to be non-uniform, a high concentration being seen adjacent to the epiphysial plate and also on the surfaces of the bone. The evidence suggested that a two-fold mechanism resulted in the uneven distribution of 99mTc-MDP. The first factor probably represented the regional distribution of blood flow with a transcapillary movement of the tracer from the capillary bed to the extravascular space; the subsequent incorporation of the tracer into bone appeared to depend on the nature of the bone matrix.


A possible mechanism for island formation by rat ascites hepatoma cells with special reference to the function of aggregation factor at the cell surface. Two tumor cell-aggregation factors of glycoprotein nature, separated from rat ascites hepatoma AH136B cells (forming cell islands in vivo), had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. The unabsorbed factor induced aggregation (as shown in the form of simple apposition) of rat ascites hepatoma AH109A cells (present as a free form in vivo) and cell adhesiveness characterized by well-defined tripartite junctional complexes, including intermediate junctions, desmosomes, and tight junctions. In contrast, the absorbed factor from AH136B cells, AH109A cells or normal rat serum aggregated AH109A cells but failed to develop the junctional complexes; only simple apposition was observed. AH109A cells themselves contained the absorbed factor but not the unabsorbed factor. AH136B cells proliferating in the skin developed the junctional complexes, while AH109A cells proliferating in the skin did not from any junctional complexes.


[Tumor prevention by vitamin A: in vitro studies with human cultured fibroblasts (author's transl)]. Following the well established prevention of chemical carcinogenesis by vitamin A in several species of laboratory animals, we performed in vitro studies with human diploid fibroblasts in culture. Vitamin A-palmitate, alltransretinoic acid and the analogue compound Ro 10-9359 were found to reduce the formation of active intermediates following the administration of G3H Benzo(a)pyrene to the cells. This effect which lead to a considerable decrease of alkylated DNA is not based on a direct inhibition of Benzpyrene metabolizing enzymes by the retinoids but by a preferential inhibition of the de novo synthesis of these enzymes. This caused the well known substrate mediated enzyme induction of benzpyrene metabolizing enzymes to cease. From our data we conclude that the mechanism of the cancer protective effect of vitamin A with respect to certain carcinogens is based on an inhibited activation of procarcinogens. This effect can also be expected in human tissues.


[Permeability of N,N-bis(2-chloroethyl)-diamido-phosphoric-acid into tumor cells (author's transl)]. The uptake of tritiated N,N-bis(2-chloroethyl)-diamido-phosphoric-acid into Ehrlich-Ascites-Tumor cells of mice was studied by means of the siliconoil-filtration technique. At 10 mM concentration no permeation of the metabolite into the tumor cells could be found within 5 min at 1 degrees C, while its congenors cyclophosphamide and 4-hydroperoxycyclophosphamide (1 mM) were shown to permeate into the cells very easily reaching saturation values. Thus lack of permeation into tumor cells of N,N-bis(2-chloroethyl)-diamidophosphoric-acid seems to be the reason for the poor cytotoxic activity of this metabolite of cyclophosphamide.


Interstitial cell carcinomas of the testis in Balb/C male mice ingesting methoxychlor. Balb/c and C3H strains male and female mice ingested 750 ppm methoxychlor or 100 ppm DDT in the diet for 2 years. Balb/c strain male mice ingesting methoxychlor developed a highly significant incidence of interstitial cell carcinomas of the testis. Balb/c strain male mice ingesting DDT and C3H strain male mice receiving methoxychlor or DDT did not have testicular tumors. The carcinomas of the testis varied from well-differentiated to poorly differentiated and undifferentiated and were capable of metastasis. Carcinomas of the testis have been described in Balb/c strain male mice, but not C3H, given estrogens. The carcinogenicity for testis of Balb/c strain male mice is most likely related to the estrogenic activity of methoxychlor.


Intermittent combination chemotherapy with chlorambucil and prednisone of low-grade malignant non-Hodgkin's lymphomas according to the Kiel classification. Fourty-three of 61 patients suffering from low-grade malignant non-Hodgkin's lymphomas according to the Kiel classification were subjected to an intermittent combination chemotherapy with chlorambucil and prednisone. In 12 of 35 evaluable patients a complete remission, in 14 of 35 a partial remission could be achieved. The mean remission time of patients with a complete remission exceeds 16 months, that of patients with a partial remission amounts to more than 8 months. In 9 of 35 cases a remission could not be achieved. As 10 of the 12 patients in whom a complete remission could be obtained are alive, the median survival time in cases of complete remission cannot yet be determined. The hematological toxicity was very low, nausea and lack of appetite were observed in 9 of 35 patients. Three of 35 treated and evaluable patients died, viz., 1 patient with a centroblastic-centrocytic lymphoma and 1 patient with a chronic lymphocytic leukemia died from the sequelae of progression during the chemotherapy of second choice according to the COP regimen. The third patient with a centroblastic-centrocytic lymphoma died from the sequelae of an intrahepatic cholestatic icterus, also during the chemotherapy of second choice according to the COP regimen. In the last-mentioned case, autopsy also confirmed a continuous complete remission of the centroblastic-centrocytic lymphoma.


The presence and significance of the Thomsen-Friedenreich antigen in mammary gland. II. Its topochemistry in normal, hyperplastic and carcinoma tissue of the breast. Normal tissue as well as various benign and malignant lesions of the breast were histochemically examined for the presence of the Thomsen-Friedenreich (TF)-antigen. Fluorescein- or 3H-labelled peanut agglutinin was used for this purpose, a lectin that is known to have a high affinity for the TF-antigen. The occurrence of this TF-antigen seemed in all cases, even in the carcinoma lobulare in situ that is regarded as being derived from myoepithelial cells by some authors, to be associated with a secretory condition. Its presence (free and neuraminic acid covered) in normal, hyperplastic and malignant breast tissue, however, cannot be considered a specific tumour associated antigen as has been previously assumed. Furthermore the investigations have shown that the intensity of fluorescence for peanut agglutinin (PNA)-receptors was generally stronger in differentiated carcinomas than in undifferentiated carcinomas of the breast. The histochemical findings are discussed with regard to diagnostical and immunotherapeutical aspects.


Iatrogenic vascular injuries. A review of 125 cases (excluding angiographic injuries). From the files of an insurance company and from a number of our own files as expert medical witness to the court we have been able to collect 125 cases of malpractice suits following a iatrogenic vascular injury. An analytic study has revealed the following: --12 vascular injuries occurred after stripping of varicose veins of the lower extremities; --4 accidents occurred during arterial surgery; --35 lesions followed orthopedic surgery; and --2 general surgery. Besides these 53 injuries following surgical procedures, 72 injuries are described following non-surgical diagnostic or therapeutic procedures: --sclerotherapy of varicose veins or angiomas: 34 cases; --injection of anesthesic agents: 20 cases; --injections, infusions, catheterisms: 16 cases; and --endoscopic procedures: 2 cases. Vascular risk exists in almost all medico-surgical procedures and requires constant and careful attention since professional responsibility is easily involved. Early recognition and early correction of the vascular lesion reduce the incidence and the gravity of the complications.


Peroperative flow and pressure measurements in profundaplasty. In twelve patients, the fluid resistance across the origin of the profunda femoris artery was assessed by flow and pressure recordings, before reconstructive profundaplasty was performed. There was a considerable range of resistance values obtained and in the eight patients having repeat flow and pressure recordings following the reconstruction in only five patients was there a reduction in the fluid resistance. This suggests that not all atheromatous plaques at the profunda femoris origin produce a blood flow disturbance to warrant profundaplasty, and may explain the variable results reported following this operation.


Intracardiac repair of tetralogy of Fallot in adults. This study demonstrates that total surgical correction in tetralogy of Fallot is a gratifying procedure even with advancing age especially when one reckons that a great majority of these patients showed pronounced disability at the time of surgery without the benefit of previous palliative procedures. However, there are technical difficulties in accomplishing repair in the fibrotic hearts of older patents. The early mortality and long term benefits in this age group closely parallel those in our over all experience with surgery for this anomaly. There was a 12.5% early and 10% late mortality in this series. Surviving patients have been followed up for a period ranging from 1 to 10 years (mean 18 months) and are found to be leading normal active lives.


Differential effects of inhibition of polyamine biosynthesis on cell cycle traverse and structure of the prematurely condensed chromosomes of normal and transformed cells. The objective of this study was to determine the points in the cell cycle at which normal and transformed cells become arrested as a result of polyamine deprivation. Treatment of normal (human fibroblast line PA2 and mouse 3T3) and transformed (CHO, HeLa and SV3T3) cells with methylglyoxal bis-(guanyl-hydrazone) resulted in a significant decrease in the levels of spermidine and spermine which was associated with an inhibition of growth. Examination of the prematurely condensed chromosomes (PCC) of the polyaminedepleted cells, revealed that normal fibroblasts were preferentially arrested in early G1 phase while a majority of cells in the transformed lines were blocked in S phase. A close examination of the PCC of the transformed cells indicated a significant decrease in the number of DNA replication sites suggesting that polyamines have an important role in DNA chain initiation.


The importance of ornithine as a precursor for proline in mammalian cells. Ornithine aminotransferase catalyzes the reversible transamination of L-ornithine to delta1-pyrroline-5-carboxylate, the immediate precursor of proline. The direction and flux through this pathway in mammalian cells has not been established. Glutamate has generally been considered to be the most important precursor for proline biosynthesis, but recent studies in xiphoid cartilage indicate that a significant fraction of cellular proline is derived from ornithine. Using newly isolated mutant Chinese hamster ovary cells with defined defects in the proline biosynthetic pathways, we now have established that cells can grow at a maximal rate with ornithine as the sole source of proline. Furthermore, we have measured the rate of proline formation from ornithine (1.6 nmol/h/10(6) cells); Future studies with these mutant Chinese hamster ovary cells may offer insight into the regulatory mechanism which coordinates proline biosynthesis from ornithine and glutamate.


Popliteal artery thrombosis in a young woman, secondary to bony exostosis. Idiopathic thrombosis of the popliteal artery in young people is exceedingly uncommon. Previously suggested etiologies are cystic adventitial disease, popliteal entrapment syndrome, Buerger's Disease, juvenile arteriosclerosis and synovial cysts. An instance of popliteal artery thrombosis secondary to compression by a bony exostosis is recorded to call attention to this unusual etiological mechanism in a 21 year old woman. Restoration of arterial continuity was achieved by endarterectomy and a venous patch graft following excision of the bony exostosis.


Serum-dependent regulation of alpha-aminoisobutyric acid uptake in bovine granulosa cells. The removal of serum from the medium of ovarian granulosa cells in exponential or confluent stages of growth results in a rapid and pronounced decrease in the rate of transport of the non-metabolizable amino acid, alpha-aminoisobutyric acid. This decrease is rapidly and completely reversed by the addition of serum. The decrease and its reversal are insensitive to inhibitors of RNA and protein syntheisis and are unaffected by a number of other metabolic inhibitors. The serum requirement cannot be replaced by peptide hormones known to stimulate cell division and secretion by these cells. These data are consistent with a model of post-translational control of AIB transport by a high-molecular-weight component of serum.


Ordered replication of DNA sequences: synthesis of mouse satellite and adjacent main band sequences. The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase, It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.


A comparative study of glycosaminoglycans in cultures of human, normal and malignant glial cells. The glycosaminoglycans (GAG) of human cultured normal glial and malignant glioma cell lines were studied using 35S-sulphate or 3H-glucosamine as markers. 35S-labelled GAG were assayed by precipitation with cetylpyridinium chloride; 3H-labelled sulphated GAG and 3H-labelled hyaluronic acid were quantitated after separation on a DEAE-cellulos column. The net production of GAG and the distribution, composition and turnover of GAG were similar in all of the normal cell lines tested, but showed a great variability in the malignant cell lines. Most of the glioma cell lines produced more hyaluronic acid and less sulphated GAG than the normal cell lines, but exceptions were noted. The GAG of the trypsin susceptible (pericellular pool of normal glial cells consisted mainly of heparan sulphate with only minor amounts of other GAG. The analogous material of most glioma cells showed hyaluronic acid as the major GAG. Material liberated by trypsin from EDTA-detached cells (membrane fraction) was enriched in heparan sulphate as compared to the entire pericellular pool. Substrate attached material (SAM) left with the plastic dish after EDTA treatment of normal cultures was rich in heparan sulphate, whereas SAM of glioma cells lacked heparan sulphate or showed greatly reduced amounts of this component. Release of newly synthesized GAG to the extracellular medium was a rapid process in the normal cells but was more or less delayed in the glioma cells. The extracellular medium of the malignant glioma cultures was consistently poor in dermatan sulphate, as compared to that of normal cultures.


Fast and slow fractions of K+ flux in human lymphocytes. Potassium influx and efflux were studied in human peripheral blood lymphocytes equilibrated over a wide range of external K+ levels. The absence of a net ion movement throughout the flux study was established, trapped space was measured with polyethylene glycol, and cells were separated from incubation media without exposure to any washing solution. There are both rapid and slow cellular fractions of 42K influx and efflux, and half-times of exchange of around 2 minutes, and 400 minutes, respectively. The rapid component is identical in magnitude to the smaller non-saturable component of cell K+, while the slow component is identified with the larger, sigmoidal, saturable component of cell K+ that was previously shown to follow a cooperative adsorption isotherm. These results support the association-induction hypothesis, which predicts (a) a rapid fraction of K+ flux due to equilibration of ion within cell water existing in a state of polarized multilayers, and (b) a slower component of K+ flux limited by adsorption onto, or desorption from, fixed anionic sites existing throughout the cell. K+ influx, as a function of external K+, showed a triphasic relation with a peak around 1 mM K+ex, then a trough around 4mM K+ex, and then a gradual rise. This relation was readily explained, in terms of the association-induction hypothesis, by the cooperative interaction between, and ion occupancy of, fixed anionic sites that adsorb K+ or Na+.


Selective suppression of the transcription of ribosomal genes in mouse-human hybrid cells. Mouse-human hybrid cells that preferentially segregate either mouse or human chromosomes were analyzed for their relative content of mouse and human rRNA genes and for their capacity to transcribe these genes. A distinctive Hind III restriction fragment containing 28S rRNA sequences was used to distinguish between mouse and human rDNA and a set of distinctive loop structures in the 45S pre-rRNA was used to distinguish between mouse and human gene transcripts. Our results indicate that the genes of only one species are transcriptionally active in these hybrid cells, even though both sets of genes are present.


Characterization of cell lines showing growth control isolated from both the wild type and a leucyl-tRNA synthetase mutant of Chinese hamster ovary cells. The genetic approach to the problem of cellular growth control is limited by the availability of recessive mutations in cell lines which are capable of growth control in vitro. The CHO cell line has yielded many recessive mutations including, for example, tsH1, a temperature sensitive leucyl-tRNA synthetase mutant, which under non-permissive conditions rapidly shuts down protein synthesis and generates uncharged tRNA. Both CHO and tsH1 are transformed, however, and do not respond to environmental stimuli with the coordinated regulation of macromolecular processes observed in normal diploid fibroblasts. We describe here the isolation and characterization of growth control revertants obtained from both CHOwt and tsH1. The best of these GRC+L-73, isolated from tsH1, had 20 chromosomes, one less than tsH1, had normal fibroblastic morphology, would not grow in suspension, required high serum concentrations for growth, grew to relatively low cell densities at saturation in monolayer culture and showed a stationary phase characterized by arrest in a G1-like state with maintenance of high viability for several weeks. It is expected that this line as well as a ts revertant GRC+LR-73 will greatly facilitate the genetic investigation of growth control and, in particular, will help to elucidate the role of uncharged tRNA in the regulation of macromolecular synthesis in mammalian cells.


Growth of human embryonic fibroblasts at clonal density: concordance with results from mass cultures. In the past, it has been difficult to grow human diploid fibroblast cells at clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1) population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non-dividing cells. In all cases essentially the same results were obtained for cultures at clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture.