Purification and properties of avian liver p-hydroxyphenylpyruvate hydroxylase.

Avian liver p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) was purified to a 1000-fold increase in specific activity over crude supernatant, utilizing a substrate analogue, o-hydroxyphenylpyruvate, to stabilize the enzyme. The preparation was homogeneous with respect to sedimentation with a sedimentation velocity (s20,w) of 5.3 S. The molecular weight of the enzyme was determined to be 97,000 +/- 5,000 by sedimentation equilibrium, and the molecular weight of the subunits was determined to be 49,000 +/- 3,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis revealed heterogeneity of the purified enzyme. The multiple molecular forms were separable by isoelectric focusing, and their isoelectric points ranged from pH 6.8 to 6.0. The amino acid compositions and tryptic peptide maps of the three forms isolated by isoelectric focusing were very similar. The forms of the enzyme had the same relative activity toward p-hydroxyphenylpyruvate and phenylpyruvate. Conditions which are known to accelerate nonenzymic deamidation of proteins caused interconversion of the multiple molecular forms. Iron was the only transition metal found to be associated with the purified enzyme at significant levels. The amount of enzyme-bound iron present in equilibrium-dialyzed samples was equivalent to 1 atom of iron per enzyme subunit. Purification of the enzyme activity correlated with the purification of the enzyme-bound iron. An EPR scan of the purified enzyme gave a signal at g equal 4.33, which is characteristic of ferric iron in a rhombic ligand field.


Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.


Isolation and characterization of tissue-specific isozymes of glucosephosphate isomerase from catfish and conger.

In teleosts glucosephosphate isomerase exists as two tissue-specific isozymes. Most tissues contain the more acidic liver-type isozyme, while white muscle contains the more basic isozyme; and a few tissues contain both the liver- and muscle-type isozymes as well as a hybird. The isozymes were isolated from catfish liver and muscle and from conger muscle and shown to be homogeneous by polyacrylamide gel electrophoresis, isoelectric focusing, analytical ultracentrifugation, and rechromatography. Both isozymes are of molecular weight 132,000 (S020,w = 7.0 S) and composed of two subunits of Mr approximately 65,000. The muscle and liver isozymes were shown to have distinct isoelectric points (catfish liver = 6.2; muscle = 7.0) and amino acid compositions. Tryptic peptide maps, after S-carboxymethylation and carbamylation, revealed several distinct differences in the primary structures of the isozymes. Although the isozymes could also be distinguished on the basis of their stabilities, most of their basic catalytic properties were found to be similar. A conger was obtained which was heterozygous for the variant allele at the muscle-glucosephosphate isomerase locus. A comparison of the variant conger muscle isozyme with the wild type revealed a single altered peptide, suggesting a point mutation. The structure-function studies, as well as the genetic studies, clearly establish that the two types of isozymes are of independent genetic origin.


Purification from human plasma of a heparin-released lipase with activity against triglyceride and phospholipids.

A triglyceride lipase different from lipoprotein lipase, but measurable only after intravenous heparin injection, has been isolated from human plasma by sequential use of heparin-Sepharose and concanavalin A-Sepharose affinity chromatography. Using these procedures, phospholipase A1 activity was found to chromatograph identically with the triglyceride lipase. The constancy of the ratio of activities after isoelectric focusing (pI 4.1) and during thermal deactivation indicates that this enzyme has hydrolase activity against both triglycerides and phospholipids. This conclusion was supported further by the homogeneity of the protein as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.


Equilibrium dialysis and cell binding studies on Bandeiraea simplicifolia lectin.

Equilibrium dialysis studies on the binding of the blood group B-specific Bandeiraea simplicifolia lectin to methyl alpha-D-galactopyranoside demonstrated the existence of one carbohydrate binding site per subunit for the tetrameric protein, with an intrinsic association constant of 8.6 x 10(4) (M) (-1) at 2 degrees and 3.3 x 10(4) (M)(-1) at 20 degrees. These values correspond to a free energy of binding, ΔG(0') (pH 7.2), of -6.21 kcal/mol and-6.06 kcal/mol at 2 degrees and 20 degrees, respectively. The sites appeared homogeneous and noninteracting. B. simplicifolia lectin receptor sites per human B erythrocyte varied from 0.72 x 10(5) to 1.34 x 10(5) with an apparent association constant of 1.1 x 10(7) (M)(-1) to 2.9 x 10(7) (M)(-l). The binding characteristics of B. simplicifolia lectin are compared to other purified lectins.


Structure of heparin. Characterization of the products formed from heparin by the action of a heparinase and a heparitinase from Flavobacterium heparinum.

The total degradation of heparin by the joint action of a purified heparinase and a heparitinase from Flavobacterium heparinum is reported. The heparinase acts directly upon heparin, yielding 52% of a trisulfated disaccharide (O-(alpha-L-ido-4-enepyranosyluronic acid 2-sulfate)-(1leads to 4)-2sulfoamino-2-deoxy-D-glucose 6-sulfate) and 40% of a tetrasaccharide besides small amounts of hexa- and disaccharides. The tetrasaccharide is in turn completely degraded by the heparitinase, forming trisulfated disaccharide and disulfated disaccharide (O-(alpha-D-glyco-4-enepyranosyluronic acid)-(1leads to 4)-2-sulfoamino-2-deoxy-D-glucose 6-sulfate) in equal amounts. These and other results indicate that the tri- and disulfated disaccharides are linked alternately, in a proportion of 3:1, respectively. The primary structure of heparin and the mode of action of the heparinase and the heparitinase are proposed based on the analysis of the different products formed by the action of the enzymes.


The stereochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate-6-phosphate aldolase.

In a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P as catalyzed by 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) of Pseudomonas putida, C--C synthesis occurred appreciably faster than C--3H bond breaking. Since tritium is present in tritiated pyruvate in tracer amounts, this result showed hydrogen isotope discrimination in pyruvate deprotonation and suggests enolpyruvate generation to be at least partially rate-limiting in the condensation reaction. Consequently, in a condensation reaction between [3-3H, 2H,H]pyruvate of known chirality and D-glyceraldehyde-3-P, the newly synthesized C--C bond would be enriched for at what was the C--H bond of chiral pyruvate, discriminating against the C--2H and C--3H bonds. Additional studies showed that condensations between (3S)-[3-3H, 2H,H]- or (3R)-[3-3H, 2H,H]pyruvate and D-glyceraldehyde-3-P yielded predominantly (3S)- or (3R)-2-keto-3-deoxy[3-3H, 2H]gluconate-6-P, respectively. By comparison with sterochemical models, it was concluded that condensation occurred with retention of configuration at C-3. Thus in the turnover of substrates as catalyzed by this enzyme, both the exchanging proton from water and D-glyceraldehyde-3-P attack the same face of the enzyme-bound pyruvyleneamine.


The sterochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate 6-phosphate and 2-keto-3-deoxygalactonate-6-phosphate aldolase of Pseudomonas saccharophila.

In Pseudomonas saccharophila 2-keto-3-deoxygalactonate-6-P aldolase (EC 4.1.2.21) is induced by growth on galatose while 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) is constitutive. These enzymes catalyze identical reactions except for the configuration fixed at C-4 during the condensation reaction. It was found with each enzyme that in a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P, the respective condensation products were formed 8 to 10 times faster than tritium was released to water. Since pyruvate deprotonation is obligatory for condensation, the above result requires a hydrogen isotope effect in enolpyruvate formation, which must be then at least partially rate limiting for C--C synthesis. Further, condensation between D-glyceraldehyde-3-P and (3R)-[3-3H, 2H,H]pyruvate or (3S)-[3-3H, 2H,H]pyruvate, as catalyzed by each enzyme, enriched for (3R)- and (3S)-3-3H, 2H-labeled condensation product, respectively. Thus, each enzyme catalyzes C--C and C--H synthesis with retention of configuration at C-3. This shows that the active sites of both enzymes are asymmetric since solutes can only approach a single face of the bound pyruvyl enolate. In addition, the respective aldehyde specific portions of the two active sites must have opposite chiralities, with respect to each other, for correctly orienting the carbonyl faces of the incoming D-glyceraldehyde-3-P, to generate the correct configuration at C-4 of the respective condensation products.


Charge effects in the activation of adenylate cyclase.

Polycations, including ribonuclease A, ribonuclease S protein and peptide, spermine, spermidine, and polylysines, enhance unstimulated and stimulated adenylate cyclase activity of beef thyroid membranes at low concentrations and inhibit these activities at high concentrations. Peak polylysine stimulation occurs with degrees of polymerization of 6 to 14, and for large polymers a potency limit for this maximum is reached at 4 X 10(-5) M expressed as lysine residues. Both enhancement and inhibition appear to be due to charge-charge interactions and are abolished by KC1. Polyanions are inhibitory only. The biphasic effect of polycations is seen on basal cyclase activity, occurs with prostaglandin E1- and 5'-guanylyl-imidodiphosphate-stimulated cyclase, but is most striking with thyrotropin. There is little enhancement of F--activated cyclase. The enhancement is not sensitive to changes in pH, Mg2+, or regenerating system and does not correlate with the stability constants between polycations and ATP. We suggest that the polycation effect is a general, electrostatic effect on membrane conformation and is not restricted to a particular receptor domain.


Biosynthesis and characterization of lipid-linked sugars and glycoproteins in aorta.

A particulate enzyme from bovine aorta catalyzes the incorporation of mannose from GDP-D-[14C]mannose into three products as follows: 1. Most of the radioactivity which is incorporated in short term incubations is into a product that is soluble in CHCl3/CH3OH (2/1, v/v). This product was purified by chromatography on DEAE-cellulose and Sephadex LH-20. The purified glycolipid was stable to alkaline saponification but released [14C]mannose when subjected to mild acid hydrolysis (1/2 = 7 min at 100 degrees in 0.01 N HCl). The purified glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did authentic dolichyl mannopyranosyl phosphate. The synthesis of the 14C-mannolipid was reversed by the addition of GDP and Mg2+. 2. [14C]mannose is also incorporated, although at a slower rate into products which are soluble in CHCl3/CH3OH/H2O (1/1/0.3, v/v). When the 1/10.3 soluble material was chromatographed on Avicel plates, it gave rise to three distinct radioactive bands which appear to be lipid-linked oligosaccharides. Mild acid hydrolysis of the 1/10.3 soluble material released water-soluble, neutral 14C-oligosaccharides which eluted from Sephadex G-50 in two or three peaks between the standards cytochrome c and GDP-mannose...


Experiments in marine biochemistry. Homarine metabolism in Penaeus duorarum.

A fractionation procedure has been developed which permits the isolation of 1 to 2 mg of homarine from a single shrimp. This procedure was used to show that homarine is endogenously synthesized by Penaeus duorarum in the free unbound form, and to study the metabolic precursors involved. Injected DL-[14C]tryptophan was not converted to [14C]homarine. However, [6-14C]quinolinic acid, a known catabolite of tryptophan, is an effective precursor. [2-14C]Acetate and [U-14C]glycerol are effectively converted to [14C]homarine while [14C]bicarbonate is poorly utilized. The injection of L-[U-14C]aspartate resulted in labeled homarine, but the quantity converted was less than expected. Since [14C]glycerol is an effective precursor there is a possibility that quinolinic acid may be formed in P. duorarum by a condensation similar to that of glyceraldehyde 3-phosphate with aspartic acid or a closely related metabolite. It is suggested that decarboxylation of quinolinic acid gives rise to picolinic acid which is methylated to yield homarine. L-[methyl-14C]Methionine efficiently provides the N-methyl carbon presumably via S-adenosylmethionine.


Omega-cyclohexyl fatty acids in acidophilic thermophilic bacteria. Studies on their presence, structure, and biosynthesis using precursors labeled with stable isotopes and radioisotopes.

Omega-Cyclohexyl undecanoic acid and omega-cyclohexyl tridecanoic acid were found in 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs. These unusual fatty acids were found in the esterified form in glyceride type complex lipids and constituted 74 to 93% of the total fatty acids in the bacteria. The fatty acids other than omega-cyclohexyl fatty acids found were 14-methyl hexadecanoic acid (3 to 15%) and 15-methyl hexadecanoic acid (1 to 6%), and trace amounts of straight chain and methyl-branched tetra- and penta-decanoic acids. Biosynthesis of omega-cyclohexyl fatty acids increased with increase in the concentration of glucose in the culture medium. The metabolism of omega-cyclohexyl fatty acids was studied using deuterium-labeled precursors by mass fragmentation analysis. The deuterium of [2-D]glucose was specifically incorporated into position 2 of the cyclohexyl ring of the fatty acids, indicating that the ring was synthesized from the glucose molecule. Radioactivity was efficiently incorporated into the omega-cyclohexyl fatty acids from labeled glucose, shikimate, and cyclohexyl carboxylate. These findings indicate that omega-cyclohexyl fatty acids are synthesized with glucose through shikimic acid and probably cyclohexyl carboxylyl-CoA derivative as the intermediates.


D-amino acid aminotransferase of Bacillus sphaericus. Enzymologic and spectrometric properties.

D-Amino acid aminotransferase, purified to homogeneity and crystallized from Bacillus sphaericus, has a molecular weight of about 60,000 and consists of two subunits identical in molecular weight (30,000). The enzyme exhibits absorption maxima at 280, 330, and 415 nm, which are independent of the pH (5.5 to 10.0), and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. One of the pyridoxal-5'-P, absorbing at 415 nm, is bound in an aldimine linkage to the epsilon-amino group of a lysine residue of the protein, and is released by incubation with phenylhydrazine to yield the catalytically inactive form. The inactive form, which is reactivated by addition of pyridoxal 5'phosphate, still has a 330 nm peak and contains 1 mol of pyridoxal 5'-phosphate. Therefore, this form is regarded as a semiapoenzyme. The holoenzyme shows negative circular dichroic bands at 330 and 415 nm. D-Amino acid aminotransferase catalyzes alpha transamination of various D-amino acids and alpha-keto acids. D-Alanine, D-alpha-aminobutyrate and D-glutamate, and alpha-ketoglutarate, pyruvate, and alpha-ketobutyrate are the preferred amino donors and acceptors, respectively. The enzyme activity is significantly affected by both the carbonyl and sulfhydryl reagents. The Michaelis constants are as follows: D-alanine (1.3 and 4.2 mM with alpha-ketobutyrate and alpha-ketoglutarate, respictively), alpha-ketobutyrate (14 mM withD-alanine), alpha-ketoglutarate (3.4 mM with D-alanine), pyridoxal 5'-phosphate (2.3 muM) and pyridoxamine 5'-phosphate (25 muM).


Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake).

The complete amino acid sequence of notexin, a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The protein consists of a single chain of 119 amino acids cross-linked by seven disulfide bridges and has a formula weight of 13,578. The main fragmentation of the peptide chain was accomplished with a staphylococcal protease specific for glutamoyl bonds. A  cyanogen bromide fragment and tryptic peptides were used to align the five major staphylococcal protease peptides. The sequence was determined by Edman degradation using the direct phenylthiohydantoin method and with carboxypeptidase A. Notexin is shown to be homologous to both porcine pancreatic phospholipase A and a phospholipase A from the venom of Naja melanoleuca.


A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.


A lipid-linked oligosaccharide intermediate in glycoprotein synthesis. Characterization of [Man-14C]glycoproteins labeled from [Man-14C]oligosaccharide-lipid and GDP-[14C]Man.

Endogenous proteins of cell-free preparations of hen oviduct labeled from GDP-[14C]Man or from [Man-14C]oligosaccharide-lipid have been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under the conditions tested, a polypeptide chain of molecular weight about 25,000 was the principle acceptor for the oligosaccharide moiety of exogenous [Man-14C]oligosaccharide-lipid. The product labeled by [Man-14C]oligosaccharide-lipid appeared identical with one of three glycoproteins formed when GDP-[14C]Man was incubated with a crude membrane fraction. These three proteins (apparent molecular weight of 75,000, 55,000, and 25,000) accounted for nearly two-thirds of the [14C]mannose-labeled glycoprotein products using GDP-[14C]Man and either the crude membrane fraction or a total oviduct homogenate. Thus, all of the mannose acceptor proteins present in the oviduct homogenate appear to be membrane-bound. Analyses of the [Man-14C]glycoproteins labeled from GDP-[14C]Man in membrane fractions from hen kidney, liver, brain, and oviduct indicated that a labeled polypeptide of apparent molecular weight 25,000 was the only major protein product common to the four preparations.


The presence of covalently linked ribonucleotides in the closed circular deoxyribonucleic acid from higher plants.

Single-stranded scissions are induced in the covalently closed circular chloroplast (ct-) DNAs from peas, spinach, and lettuce plants by treatment with alkali or by incubation with a mixture of ribonucleases A and T1. These scissions are due to the presence of covalently linked ribonucleotides in these closed circular DNAs. By comparing the scission rates of these ctDNAs to the scission rate of RNA, it has been estimated that pea and spinach ctDNAs contain a maximum of 18 +/- 2 ribonucleotides/molecule, while lettuce ctDNA contains a maximum of 12 +/- 2 ribonucleotides/molecule. Further studies with pea ctDNA by electron microscopic methods have shown that pea ctDNA contains 19 alkali-labile sites at specific locations. A map of the relative positions of the alkali-labile sites has been constructed. These alkali-labile sites are presumably due to the insertion of individual ribonucleotides.


Physical and chemical characterization of purified ovalbumin messenger RNA.

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.


Nuclear deoxyribonucleic acid polymerase. Further observations on the structure and properties of the enzyme from human KB cells.

At low ionic strength KB cell DNA polymerase N1 forms large aggregates of a size comparable to those of DNA polymerase C. However, in contrast to polymerase C, the polymerase N1 aggregate: (a) retains the distinctive features of the polymerase N1 monomer, specifically its relative insensitivity to salt and to p-hydroxymercuribenzoate, and its pI of 9.3; and (b) is quantitatively converted to the polymerase N1 monomer form at appropriate ionic strength. It is important to recognize that since both polymerase N1 and polymerase C undergo salt-dependent association-dissociation reactions, attempts to distinguish these clearly indedependent polymerase species on the basis of size criteria can be very misleading. This is particularly true in relatively impure enzyme fractions that are generally isolated from eukaryotic tissue sources in low ionic strength buffers. We had earlier reported (Wang, T. S.-F., Sedwick, W. D., and Korn, D. (1974) J. Biol. Chem. 249,841-850; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1972) J. Biol. Chem. 247,5026-5033; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1974) Methods Enzymol. 29, 89-102) that DNA polymerase N1 could not utilize homoribopolymer templates. We have re-examined this question with a modified and more stringent method of product assay, and we show here that a greater than or equal 95% homogeneous preparation of polymerase N1 can copy the primer-template (A)n-(dT)-/16 at about one-half the rate that it copies activated DNA under optimum incubation conditions.


Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity.

In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself.


A crystallographic study of deoxy cobalt (II) mesoporphyrin IX myoglobin.

The crystal structures of acid metmyoglobin and deoxy cobalt(II)mesoporphyrin IX myoglobin were compared by a difference Fourier analysis at 2.5 A resolution. No large differences in protein conformation were observed. The greatest density of structural differences was found in the heme region. There was a loss of the histidine-bound sulfate ion and of the metal-bound water molecule, as well as a shift in the position of the prosthetic group with associated changes in the adjacent globin. The structural changes resulting from the substitution of ethyl for the vinyl side chains of the porphyrin were clearly observed. There was also a suggestion of a conformational change of the porphyrin ring. It was not clear whether there was any change of the metal position relative to the porphyrin plane or proximal histidine.


Treatment of intracapsular hip fractures by the Deyerle method. A comparative review of one hundred and nineteen cases.

In a retrospective review of 119 intracapsular hip fractures treated by the Deyerle method (seventy-nine patients treated at San Francisco General Hospital and forty at Brookside Community Hospital), fifty-nine patients were available for follow-up after two or more years or were rated as failures before that time. In the San Francisco General series the incidence of avascular necrosis was 43 per cent and of non-union, 23 per cent, while in the Brookside series the incidences were 12.5 per cent and 12.5 per cent, respectively. The functional end results in the San Francisco General series were rated excellent or satisfactory in 40 per cent and failures in 60 per cent, and in the Brookside series there were 75 per cent excellent or satisfactory results and 25 per cent failures.


The use of methylmethacrylate as an adjunct in the internal fixation of unstable comminuted intertrochanteric fractures in osteoporotic patients.

Forty-two unstable, four-part intertrochanteric fractures in forty-one patients (seventy-one to 104 years old) with severe osteoporosis were treated by open reduction and internal fixation (Jewett nail or compression screw-plate) supplemented with methylmethacrylate packed into the curetted medullary space. One patient was lost to follow-up, one died of a myocardial infarction at six weeks, and one was excluded because of an unsuspected myeloma found at the fracture site. All patients were sitting up in a chair the day after operation. Full weight-bearing on the limb was started within three weeks by thirty patients and at an average of 118 days by six who had very comminuted fractures. Three patients, non-ambulatory preoperatively, did not walk after operation. Of the thirty-eight fractures followed for from nine to thirty-seven months, thirty-seven healed with no loss of position. One fracture which had been fixed with the nail and cement not extending far enough into the head and neck displaced, and the operation had to be repeated, this time with a successful result. The fractures healed by periosteal new-bone formation. There was no evidence of avascular necrosis or wound complications.


Legg-Perthes disease in the young hip--does it necessarily do well?

Thirty-one patients (with forty involved hips) in whom Legg-Perthes disease developed when they were less than five years old were reviewed. Previously defined roentgenographic features of prognostic significance were measured. Long-term follow-up (average, 129 months) allowed statistical testing of factors previously thought to be of significance in predicting outcome. Many of these patients were found to have poor results as measured roentgenographically. In addition, many prognostic indications were found not to be independent measures and of those demonstrating independence, none correlated with the functional outcome.


Walking patterns of men with unilateral surgical hip fusion.

The gait of men with unilateral hip fusion is somewhat slow, asymmetrical, and arrhythmic as compared with that of normal men. Compensation for absent hip motion is accomplished by increased transverse and sagittal rotation of the pelvis, increased motion in the sound hip, and increased flexion of the knee throughout the stance phase on the fused side. Relationships between the fusion position, certain physical traits, and walking performance suggest that the best gait can be expected in young patients who have free motion of the lumbar spine, the sound hip, and the knee on the side of fusion, and who have equal limb lengths and a hip fused in a position that does not include excessive adduction.


Femorotibial weight-bearing areas. Experimental determination.

We measured the areas of the weight-bearing surfaces of the femorotibial joint of specimens subjected to a compressive force of between 225 and 250 kiloponds, which is considered a physiological load, exerted through the center of the knee. We found the areas to range from 20.13(2) to 11.60 cm2 from extension to flexion of the knee in normal knees, and from 12 cm2 to 6 cm2 after removal of the menisci.


Madelung's deformity. An end-result study of surgical treatment.

A long-term follow-up (average, eight years) of surgical treatment of thirteen cases of Madelung's deformity in eight patients showed that the Darrach procedure was successful when there was mild deformity and that biplane osteotomy of the radius combined with the Darrach procedure was needed for severe deformity. Subluxation of the lunate bone ulnarward occurred after the Darrach procedure but was not clinically symptomatic.


Anterior angulation deformity of the radial head. An unusual lesion occurring in juvenile baseball players.

Four cases of an unusual angulation deformity of the radial head were seen in juvenile baseball players, with or without associated lesions of osteochondritis in the contiguous capitellum of the humerus and loose-body formation. The established deformity did not show a tendency toward anatomical restitution. In each case, symptoms produced by this deformity were noted to appear in association with athletic activity.


Tendon and pulleys at the metacarpophalangeal joint of a finger.

In fresh frozen traumatically amputated forearms with a constant tension of one kilogram on the flexor profundus tendon and the interphalangeal joints fixed in full extension by a Kirschner wire, the excursion of the tendon at the metacarpophalangeal joint and the force at the finger tip were correlated with different angles of flexion of the joint, first with the finger intact and then after varying amounts of advancement of the metacarpophalangeal joint pulley system. Pulley advancement increased the tendon excursion required to flex this joint and thus the mechanical advantage at this joint, but only when the joint was partly flexed. The extra excursion required at the metacarpophalangeal joint would be expected to weaken the interphalangeal joints at full flexion. Advancement also permitted ulnar-radial displacement of the tendon at the level of the metacarpophalangeal joint and hence could accentuate ulnar or radial drift. Pulley advancement is not recommended.


The fascia of the distal phalanx.

The anatomy of the distal phalanx of the human finger was studied in preserved and fresh specimens and the fascia was related to associated structures as a mechanism for prehension. Particular attention was directed to the lateral interosseous ligaments of the distal phalanx and their possible function during gripping with the digital pulp.


Results of Harrington instrumentation and fusion in the adult idiopathic scoliosis patient.

Between January 1961 and December 1972, 132 patients over twenty years old with idiopathic scoliosis were surgically treated using Harrington instrumentation and fusion techniques. All patients were seen by us because of curve magnitude or symptoms secondary to scoliosis. The average correction obtained at operation was 48 per cent. Twenty-four patients had early complications. Fifty-two had late complications, the most frequent of which was pseudarthrosis. The majority of patients had significant improvement in symptoms as a result of surgical correction and stabilization.


The mechanical properties of bone tissue in children.

Specimens of femoral cortical bone from eighteen subjects between two and forty-eight years old were loaded in bending. Compared with the bone of adults, that of children had a lower modulus of elasticity, a lower bending strength, and a lower ash content. However, the children's bone deflected more and absorbed more energy before breaking. It also tended to absorb more energy after fracture had started. The typical greenstick fracture surface of many specimens of children's bone requires more energy for its production than the relatively smooth surface of adult specimens.


Pyogenic infection of the sacro-iliac joint. Report of thirteen cases.

Thirteen cases of infectious sacro-iliitis caused by pyogenic organisms are reported and compared with thirty-four cases in the literature. Staphylococcus was the commonest infecting organism (six cases). Two clinical patterns were noted, acute (nine cases) and subacute (four cases). Treatment was by antibiotics and bed rest in all patients. Surgery was indicated for diagnosis in three patients and for abscess in three patients. All of our patients had very good functional recovery.


Infection after silicone prosthetic arthroplasty in the hand.

After 2105 silicone prosthetic arthroplasties in the hands of 631 patients, infections occurred in ten. The average time from operation to onset of signs and symptoms of infection was seventeen days, and in seven of the ten patients the infecting organism was Staphylococcus aureus. Seven patients ultimately were treated by removal of the prosthesis, intravenous antibiotics for about five days, and oral antibiotics for about two weeks. At follow-up, twelve to fifty-six months after operation, nearly all of the patients had stable, somewhat stiff but pain-free joints, the results being similar to those after resection arthroplasty.


Acute renal failure following trauma.

Acute renal failure is often fatal, but usually this complication following trauma is avoidable. Of fifteen patients with acute tubular necrosis associated with severe trauma, thirteen survived. This is a marked improvement in survival rate compared with the rates previously published. We credit the improvement to aggressive medical and surgical treatment by a team of orthopaedic surgeons, nephrologists, and surgeons.


The treatment of infected non-union of fractures of long bones. Study of sixty-four cases with a five to twenty-one-year follow-up.

The result of treatment of sixty-four patients with infected ununited fractures of long bone by debridement of soft tissue and bone and open suction-irrigation drainage, followed by rigid stabilization of the non-union and cancellous bone-grafting when indicated, was bone union in sixty cases, even in the presence of infection. The late results five to twenty-one years later showed good function despite the fact that prolonged periods of hospitalization were often required. The importance of local treatment of the infection prior to stabilizing the fracture fragments, and the use of cancellous bone grafts when necessary, are emphasized.


Congenital paralytic vertical talus. An anatomical study.

Dissections of the feet of a three-month-old infant with paralytic congenital vertical talus secondary to lumbar myelomeningocele were compared with a dissection of a normal foot. The major differences appeared to be absence of the plantar intrinsic muscles and dorsal dislocation of the talonavicular joint. It is postulated that the pathological process begins as a failure of the intrinsic muscles to oppose the unbalanced, active dorsiflexion forces of the anterior crural muscles. This imbalance then allows disruption of the talonavicular joint, mechanically the least stable joint in the mid-part of the foot. All dorsiflexion forces acting on the ankle then become ineffective and plantar flexion forces serve only to pull the calcaneus and talus into equinus, causing a "vertical" talus. Treatment must be directed at reducing the talonavicular dislocation, correcting the equinus deformity of the hind part of the foot, and substituting for the undeveloped plantar intrinsic muscles.


Flat foot. A preliminary report of an operation for severe cases.

The pathogenesis of flat foot and its operative correction for severe cases are reviewed. The importance of the medial plantar fascia in maintaining the structural integrity of the foot is emphasised. Reinforcement of an incompetent plantar fascia by separating the inner half of the calcaneal tendon and attaching it to the neck of the first metatarsal has given results in three patients that were satisfactory at two, six and seven years later.


Lumbo-sacral fusion with spring fixation.

A method of posterior lumbo-sacral fusion is described in which springs are used to fix the grafts and to maintain immobilisation. Fifty patients with lumbar disc degeneration or spondylolisthesis have been reviewed. The results have been assessed clinically by grading the symptoms before and after operation. Stay in hospital has been short and return to work rapid. There has been a high proportion of good results.


Cobalt toxicity after McKee hip arthroplasty.

The significance of cobalt as a cause of symptoms after McKee hip arthroplasty is discussed. Seven patients are described in whom such arthroplasties. became unsatisfactory after periods varying from nine months to four years. Six of these patients were cobalt-positive but nickel- and chrome-negative on patch testing. Macroscopic and histological necrosis of bone, muscle and joint capsule around the prostheses was found in five patients whose hips were explored. The symptoms were progressive pain, a feeling of instability, and in two cases spontaneous dislocation. Radiological features included acetabular fracture, bone resorption, loosening and dislocation of the prosthesis. Increased cobalt concentrations (determined by atomic absorption spectrophotometry) in the urine of four patients and in a variety of tissues in one patient are presented. Patch testing is recommended in the investigation of patients with troublesome McKee hip arthroplasties


A radiological study of fractures of acrylic cement in relation to the stem of a femoral head prosthesis.

The incidence and prognostic significance of fractures of acrylic cement related to the stem of a femoral head prosthesis in total hip replacement are examined. These fractures are demonstrated when the cement has been rendered radio-opaque by the addition of barium sulphate. One and a half percent of the radiographs of 6,649 patients showed these fractures, which were sometimes associated with subsidence of the prosthesis. Fracture of the cement was usually evident at the six-month post-operative review, if it occurred at all. This radiological complication was devoid of symptoms in the majority of cases and tended to occur in patients with excellent functional recovery. In a minority of patients pain in the thigh during the first six months seemed likely to be explained by this fracture. Slight subsidence of the prosthesis in the cement bed appeared to result in a new and final position of stability. The prognosis was very good; only when separation of the fracture exceeded about 4 millimetres was the prognosis doubtful, in which case a chronic deep infection might be suspected. Possible mechanical and structural causes of fracture of the cement are discussed.


Contact pressures in the loaded human cadaver hip.

The purpose of the work described was to find the average pressure on each of several areas of the acetabular cartilage of the cadaver hip under physiological loads. By obtaining load-deflection curves for one chosen area of cartilage, firstly with all the cartilage present and then after the successive removal of other areas, the fractions of the original load carried by the several areas were found, and hence the average pressures on those areas. Seventeen hips (age range twenty-two to eighty-seven years) were examined. Local pressures varied from zero to 3.4 times the average pressure in each hip. The highest pressures in the series (about 4 to 5 megaNewtons per square metre) were on the areas of thin fibrocartilage which were identified at the zenith of certain acetabula. The results are too few to establish whether or not the pressure distribution was age-related. The higher pressures found are within the range which in other experiments has led to fatigue failure of femoral head cartilage, and it is suggested that hips in which such pressures exist under loads of three times body weight may be predisposed to osteoarthritis.


An operation for partial closure of an epiphysial plate in children, and its experimental basis.

In two cases of partial closure of epiphysial causing angular deformity, one of a finger and one of the lower end of a femur, the deformities were corrected by growth after resection of a small peripheral bone bridge and its replacement with a free transplant of fat. This procedure has a firm basis on experimental work. Thus, when part of a growth plate in the rabbit is destroyed but the formation of a bone bridge uniting epiphysis and metaphysis is prevented, the destroyed part is replaced by regeneration of cartilage from the adjacent parts of the plate. Moreover, it has been proved in rabbits that when a bone bridge is induced and later resected and replaced with fat, recurrence of the bridge can be prevented and subsequent growth may correct the deformity.


The impact of arthroscopy on the management of disorders of the knee.

A study has been made of 800 consecutive arthroscopic examinations of the knee with special emphasis on the changes in management that resulted. Of 614 knees that would have been operated upon if arthroscopy had not been available, open operation was avoided in 32 per cent, a defferent operation was done or planned in 27 per cent, and there was no important change in the remaining 41 per cent. Of 186 knees that would have been treated conservatively if arthroscopy had not been available, the examination resulted in some immediate benefit to 30 per cent of the patients. There were no wound infections after arthroscopy alone, and the known incidence of incorrect interpretation was 1.4 percent.


The diagnosis of problems after meniscectomy.

The causes of persistent symptoms after meniscectomy have been assessed clinically, radiologically and arthroscopically in 174 knees. The commonest finding was early degenerative arthritis (chondromalacia) of the femoral condyle (40 per cent). Retained fragments of meniscus were less common than expected (13 per cent), and lesions of the other meniscus were rare (5 per cent). The clinical diagnosis was altered in 42 percent by arthroscopic examination. Arthroscopy was found to be a valuable technique in this group of patients with problems of diagnosis.


The pathological basis for skeletal scintigraphy.

Skeletal scintigraphy, which has now been established as a useful and accurate method of detecting early skeletal metastases and assessing their response to treatment, has been investigated for its pathological basis. Histological examination of several hyndred necropsy specimens, from sixty-eight patients who died from malignant disease, showed a significant increase of osteoid and immature woven bone in the presence of metastatic cancer. Tumour-cell suspensions of the VX2 carcinoma were injected into the medullary cavity or on to the periosteal surface of the ilia or tibiae of New Zealand white rabbits. A combination of bone destruction and new bone formation, similar to the autopsy material, was seen. There were at least two mechanisms for the new bone production. Initially, intramembranous ossification was seen in the fibrous stroma surrounding the tumour. Once the cortex was involved and cortical bone destruction had occurred, large amounts of woven bone resembling fracture callus were laid down. The new bone had a markedly increased avidity for bone-seeking isotopes, indicating why skeletal scintigraphy was useful. A further twenty rabbits, in whose ilia the VX2 carcinoma was growing, were treated by local irradiation. When treatment was successful the tumour was destroyed, the production of new bone ceased, and the lesion lost its increased avidity for bone-seeking isotopes, indicating that skeletal scintigraphy could be used to assess the response of skeletal metastases to therapy.


Intraosseous hypertension and pain in the knee.

The intraosseous pressure in the femur and tibia near the knee and in the internal saphenous vein at knee level was measured in fifty-three patients with suspected knee lesions. There were four groups: with and without degenerative osteoarthritis and with and without aching rest pain of the knee region. Low intraosseous pressures were found in patients with neither osteoarthritis nor rest pain, and in half the patients with osteoarthritis but without rest pain. Low pressures were found in the tibia, but very high intraosseous pressures were found in the femur in most patients with osteoarthritis and rest pain. Patients with no osteoarthritis but with rest pain mostly had high pressures in both the tibia and the femur.


Spinal lesions in battered babies.

The case histories of five young children are presented to demonstrate the radiological appearances of trauma to the spine thought to be the result of maltreatment by either parent or guardian. Four of the children were admitted to hospital within a period of three years, all being under the age of two years.


Nievergelt-Pearlman syndrome with impairment of hearing. Report of three cases in a family.

Three cases of the Nievergelt-Pearlman syndrome occurring in a family are presented. In addition to the major features of the syndrome (symphalangism, carpal fusion, atypical club foot with massive tarsal fusion, and congenital dislocation of the head of the radius), all three patients exhibited conductive deafness, which had not been recorded in previous reports. The clinical findings strongly suggested that the loss of hearing was due to fusion of the ossicles of the middle ear.


Lesions of bone in disseminated infection due to the mycobacterium avium-intracellulare group. Report of a case.

A white child with a long history of illness from the age of six was thought at first to have Hodgkin's disease. There followed an acute illness with lesions involving glands, lungs, bone and skin. Mycobacterium avium-intracellulare group (Battey) was isolated from various lesions at the age of thirteen. After six years of continuous treatment the patient, now eighteen, is living a normal life.


The incidence and pattern of knee injury associated with dislocation of the hip.

A retrospective survey of 135 posterior dislocations and fracture-dislocations of the hip was carried out in order to define the pattern of associated knee injuries. Thirty-five patients had sustained a significant injury to the knee, of which twenty-five were clearly attributable to a direct blow on the front of the knee (fractured patella, traumatic chondromalacia, fractures of femoral and tibial condyles) and ten were compatible with valgus, varus or rotational forces (medial, lateral and cruciate ligament tears). The second type of injury has not been widely recognised but it is important that it should not be overlooked.


False aneurysm of femoral artery from cancellous exostosis of femur. Report of a case in a boy of fourteen.

A boy aged fourteen trapped and tore at its origin a branch of the terminal part of the superficial femoral artery over a cancellous exostosis during extension of the knee from the position of full flexion. Three months later an expanding false aneurysm necessitated arteriography, after which suture of the tear gave a successful result.


Superficial radial neuropathy.

Isolated neuropathy of the cutaneous branch of the radial nerve is a rarely recognised condition. Five cases were described in 1932 by Wartenberg, who suggested the name cheiralgia paraesthetica. The condition has also been described as Wartenberg's disease. Twelve cases of isolated neuropathy of the cutaneous branch of the radial nerve are described, the literature is reviewed and the clinical picture outlined. In six of the cases the condition subsided without treatment, in two there was a good response to local injection  of hydrocortisone, and in four cases a satisfactory result followed resection of the nerve. The course and distribution of the superficial branch of the radial nerve are described. The need to avoid the nerve during operations around the wrist is stressed.


Dorsal dislocation of the first metatarso-phalangeal joint. Report of four cases.

The anatomy of the first metatarso-phalangeal joint and of dorsal dislocation of the phalanx are described. As similar lesions in the hand, closed reduction is impossible because of interposition of the volar plate. Open reduction is essential and should be performed as soon as possible after the injury.


Bone and joint involvement in primary amyloidosis.

Primary amyloidosis is an interesting clinical entity in which amyloid is deposited in various organs, particularly mesodermderived tissues such as heart, skeletal muscle, skin, connective tissue and bone. A case with multiple lytic bone lesions is presented. Comparison to previous similar cases is made with attention being directed to the typical distribution of lesions about large joints with associated soft-tissue prominence and increase in the articular space. The differentiating radiologic features are compared to those of rheumatoid arthritis, hyperparathyroidism, and lytic metastatic lesions, with particular attention being given to the osteolytic lesions of plasmacytomas associated with multiple myeloma.


The radiologic demonstration of early changes in ulcerative colitis by double contrast technique.

The double contrast technique can be applied safely to the investigation of patients with suspected inflammatory bowel disease. The early changes of mucosal hyperemia and edema can be detected when the colon has been adequately cleansed. The extent and distribution of disease can be mapped with precision. This is of considerable importance in the differential diagnosis between ulcerative and granulomatous colitis and in the choice of appropriate treatment. Even in early stages of the disease there is a high correlation between the radiographic abnormalities and the endoscopic findings.


Angiographic features of squamous cell carcinoma of the pancreas.

The angiographic findings in two cases of squamous cell carcinoma of the pancreas are reported. Tumor blushes were present in both cases and one case showed hypervascularity. These angiographic features distinguish this lesion from the typical adenocarcinoma of the pancreas. Squamous cell carcinoma should be included in the differential diagnosis when hypervascularity or a tumor blush are demonstrated in the pancreas.


"Rectal ears".

"Rectal ears," transitory protrusions of the rectum into the internal inguinal rings, were observed in a 16-month-old girl who also had "bladder ears." Inguinal hernias were not present. The etiology and significance of "rectal ears" has not been determined.


Antral infracolic volvulus of the stomach.

Two cases of antral infracolic volvulus of the stomach are presented. The etiology of gastric volvulus and the difference between the organo-axial and mesentero-axial volvulus are discussed. Further subdivision into partial or complete volvulus and the relationship between the transverse colon and the stomach during gastric volvulus is emphasized. Awareness and understanding of gastric volvulus may allow roentgen diagnosis.


Pulmonary embolic disease: the roles of angiography and lung scanning in diagnosis.

The definitive diagnosis of pulmonary embolism remains difficult despite recent advances in nuclear medicine and angiography. In 10% of a selected series of 145 patients a negative chest x-ray and a positive lung scan was associated with no arteriographic evidence of pulmonary emboli. A normal perfusion lung scan excludes significant pulmonary embolism. When pulmonary arteriography is necessary, a biplane selective pulmonary angiogram should be performed and subselective injections may be required. Pulmonary arteriography is less of a threat to a patient suspected of having pulmonary embolic disease than inappropriate treatment.


Cardiac calcification due to myocardial abscess.

Dense calcification of an old abscess of the left ventricular wall was discovered in a patient with mitral and aortic valvular disease. In spite of the fact that myocarditis occurs in a variety of settings, calcification of a myocardial focus of infection is rare and has not been reported previously.


Experience with modified remotely controlled fluoroscopic equipment for gastrointestinal examination in debilitated patients.

The concept and realization of a highly automated remotely controlled fluoroscopic system, applicable to the examination of debilitated or uncooperative patients, have been described. The addition of a rotating cradle, remotely controlled barium administration, magazine-fed roll film camera, remotely inflated paddle for prone-pressure spot filming, and a vacuum restraining device have been described for the examination of uncooperative as well as cooperative patients. These patients can be examined with as high a degree of sophistication and automation as is now available. Future developments in this area may lie in the realm of multitable remotely controlled fluoroscopic rooms, operation of the above equipment by a super technologist, programmed gastrointestinal examinations utilizing the above equipment and automated programming, and teleremote controlled fluoroscopy from a centralized location with coaxial cable or microwave transmission.


An evaluation of a high resolution closed-circuit television link in a hospital radiology department.

We have installed and developed a high resolution closed-circuit television system for transmission of radiographic film images from the emergency room in our hospital to the reading room in our radiology department. Using this system, the radiologist can consult promptly on all films and he has increased efficiency. To transmit high detail images, our television system uses a high resolution vidicon camera with a scan rate of 1029 lines and with a limiting resolution of 1200 TV lines. To further increase the overall diagnostic value of our system, we have added remote controls for changing the magnification, illumination and position of the film. In a preliminary clinical trial, seven radiologists were each shown 27 patient film sets transmitted through the television system and then viewed directly on a conventional view box. An analysis of variance of the accuracy rates in each method showed a statistical difference in methods. Even though the error rate was slightly higher using closed-circuit system, radiologist performance was of acceptable accuracy when considering the advantages of the system. In addition, many of  the factors reducing the TV system performance have been eliminated or controlled, and further experiments should reflect an even smaller difference between the two methods.


Alterations at the cellular level in the brain following repeated angiography: protective effect of corticosteroids.

Repeated injections of angiographic contrast media disrupt the blood-brain barrier and lead to transient cerebral edema in the normal experimental animal brain. The corticosteroid, dexamethasone, protects against the development of this edema without altering the effect of the contrast media on the tight junctions between adjacent endothelial cells. Indirect evidence is presented to support the theory that the corticosteroids function to maintain the integrity of the endothelial cells.


Myelographic appearance of meningo-vascular lymphoma involving cauda equina.

The myelographic pattern of diffuse meningo-vascular infiltration of the cauda equina is presented as characterized by marked enlargement of the nerve root shadows and occasional nodulation. This appearance, when coupled with appropriate clinical and laboratory findings, allows a specific diagnosis to be made.


Bilateral agenesis of internal carotid artery.

This could be the first case of bilateral agenesis of the internal carotid artery reported "in vivo." Angiogrpahic study has allowed us to recognize the anatomic details of this malformation. The absence of the two carotids is being compensated for by a trigeminal artery on the (left) side and by a hypertrophied accessory meningeal artery on the (right) side. Standard x-rays of the base of the skull (including tomography) revealed a dilation of the foramen ovale, related to the hypertrophied right accessory meningeal artery, and a complete atresia of the two carotid canals. This latter finding permits us definitely to postulate the nature of the congenital anomaly. Finally, the carotid agenesis was associated with the presence of three arterial aneurysms, situated respectively in each carotid territory and in the territory of the basilar artery.


Hypochondroplasia.

Hypochondroplasia is an autosomal dominant discrete form of short-limbed dwarfism with roentgenographic changes similar to, but distinctive from, achondroplasia. The patients are normal at birth and in early childhood present with short-limbed dwarfism, thick body build, long trunk, normal head, moderate lumbar lordosis and broad, stubby hands and feet. A high incidence of mental retardation and absence of other neurologic complications is distinctive from achondroplasia. Diagnosis of hypochondroplasia is primarily radiogrpahic. No single diagnostic radiographic sign is present, and thus, a complete radiographic survey of the whole skeleton is required for diagnosis. Pertinent radiographic findings include a normal skull, short, broad long bones with prominent bony sites of muscle attachment, normal growth plate, prominent styloid process of the ulna and lateral malleolus of the fibula, shortened base of the iliac bones, horizontal hypoplastic low-set sacrum and mild narrowing of the interpediculate distance of the lumbar spine. Differential diagnosis is from other types of short-limbed dwarfism, particularly achondroplasia. Kozlowski, an authority on hypochondroplasia, believes this form of short-limbed dwarfism, if carfully searched for, may be more common than achondroplasia.


Absence of ribosomal DNA amplification in the meroistic (telotrophic) ovary of the large milkweed bug Oncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae).

In the typical meroistic insect ovary, the oocyte nucleus synthesizes little if any RNA.  Nurse cells or trophocytes actively synthesize ribosomes which are transported to and accumulated by the oocyte.  In the telotrophic ovary a morphological separation exists, the nurse cells being localized at the apical end of each ovariole and communicating with the ooocytes via nutritive cords.  In order to determine whether the genes coding for ribosomal RNA (rRNA) are amplified in the telotrophic ovary of the milkweed bug Oncopeltus fasciatus, the percentages of the genome coding for ribosomal RNA in somatic cells, spermatogenic cells, ovarian follicles, and nurse cells were compared.  The oocytes and most of the nurse cells of O. fasciatus are uninucleolate.  DNA hybridizing with ribosomal RNA is localized in a satellite DNA, the density of which is 1.712 g/cm(-3).  The density of main-band DNA is 1.694 g/cm(-3).  The ribosomal DNA satellite accounts for approximately 0.2% of the DNA in somatic and gametogenic tissues of both males and females.  RNA-DNA hybridization analysis demonstrates that approximately 0.03% of the DNA in somatic tissues, testis, ovarian follicles, and isolated nurse cells hybridizes with ribosomal RNA.  The fact that the percentage of DNA hybridizing with rRNA is the same in somatic and in male and female gametogenic tissues indicates that amplification of ribosomal DNA does not occur in nurse cells and that if it occurs in oocytes, it represents less than a 50-fold increase in ribosomal DNA.  An increase in total genome DNA accounted by polyploidization appears to provide for increasing the amount of ribosomal DNA in the nurse cells.


BALD-2: a mutation affecting the formation of doublet and triplet sets of microtubules in Chlamydomonas reinhardtii.

The mutant strain bald-2 is unique among "flagellaless" strains of Chlamydomonas reinhardtii isolated to date, in that it possesses a mutant basal body: it is only capable of forming a ring of nine singlet microtubules, 180 nm in diameter, instead of the usual triplet basal body which is 225 nm in diameter.  This singlet basal body lacks structural stability and the ability to associate with striated fiber material but retains two critical properties of basal bodies, namely, information specifying the length to which it should elongate and the ability to induce, albeit rarely, a flagellar transition region, a short, singlet-containing axoneme, and a specialized tunnel in the cell wall through which flagella normally emerge.  The mutation seems to be specific for B- and C-microtubule synthesis or assembly since all other cytoplasmic sets of microtubules appear normal in numbers, orientation, and stability.


Large electrical currents traverse growing pollen tubes.

Using a newly developed vibrating electrode, we have explored the electric fields around lily pollen germinating in vitro.  From these field measurements, we infer that each weeted pollen drives a steady current of a few hundred picoamperes through itself.  Considered as a flow of positive ions, this current enters an ungerminated grain's prospective growth site and leaves it opposite end.  After a grain germinates and forms a tube, this current enters most of the growing tube and leaves the whole grain.  The current densities over both of these extended surface regions are relatively uniform, and the boundary zone, near the tube's base, is relatively narrow.  This current continues as long as the tube grows, and even continues when elongation, as well as cytoplasmic streaming, are blocked by 1 mug/ml of cytochalasin B.  After a otherwise indistinguishable minority of tubes have grown to lengths of a millimeter or more, their current comes to include an endless train of discrete and characteristic current pulses as well as a steady component.  These pulses are about 30s long, never overlap, recur every 60-100s, and seem to enter a region more restricted to be growing tip than the steady current's sink.  In most ways, the current through growing lily pollen resembles that known to flow through focoid eggs.


Role of microvilli in surface changes of synchronized P815Y mastocytoma cells.

The surface morphology of synchronized P815Y mastocytoma cells has been examined by scanning electron microscopy.  Early G1 cells are comparatively smooth or light villated, whereas at later stages the surface becomes progressively more villated.  In G1 cell most microvilli have a uniform diameter, whereas in S and G2 cells, many microvilli show branching and often originate from much larger surface protuberances.  Small "blebs" are seen on the surface of many cells but these structures do not appear to be a characteristic feature of cells at any one stage of the cycle.  The presence of microvilli increases the total surface of the cell to such an extent that the ratio of volume to surface area remains constant throughout the cell cycle.  The mechanism of cytokinesis is thus a physical one, involving the unfolding of previously accumulated microvilli.


Mechanisms of lysosomal enzyme release from human polymorphonuclear leukocytes.  Effects of phorbol myristate acetate.

PMA enhanced release of the azurophil granule enzyme, beta-glucuronidase, as well as lysozyme, from cytochalasin B-treated PMN's exposed to either zymosan particles or C5a.  PMA was active at nanomolar concentrations, was not toxic to the cells, and was most effective when present for brief durations (0-1 min) before exposure of the cells to the stimuli.  Beta-glucuronidase was not released in significant amounts from PMN's exposed to PMA alone, in the absence of stimuli such as zymosan or C5a.  In contrast, only the specific granule enzyme, lysozyme, was released from unstimulated cells.  Electron micrographs of cells exposed to PMA revealed an increase in the number of visible cytoplasmic microtubules as compared to control cells.  Enhancement of lysosomal enzyme (beta-glucuronidase) release by PMA appears to be independent of effects on release of specific granule enzymes (lysozyme), but rather is likely due to PMA-induced elevations of cellular cGMP.


Nucleocytoplasmic movement of fluorescent tracers microinjected into living salivary gland cells.

The permeability of the nuclear envelop of a somatic cell, the C. thummi larval salivary gland cell, was studied by intracellular microinjection of fluorescent molecular tracers.  As shown previously in oocytes (4,5,15,16), the envelop is permeable to a wide variety of materials, including molecules which are large enough to possess condiderable biological specificities and to play important roles in regulation of cellular activities.  The envelop exhibits transport selectivity on the basis of size in the range of naturally occurring intracellular materials and it may thus perform important controlling functions in nucleocytoplasmic exchange.  The nucleus to cytoplasm movement of in vivo ribonucleoprotein particulates in these synthetically active cells probably requires conformational changes in the particulates and/or the envelope pore complexes; morphological evidence exists for such processess in these cells (20).


Changes in tight junctions of thyroid epithelium with changes in thyroid activity.

The morphology of the tight junction of rat thyroid epithelium was examined in freeze-fractured material fixed in glutaraldehyde and briefly glycerinated.  In normal thyroids the overall appearance of this junctional specialization resembled that of other cell types in many respects.  Short-term changes in thyroid activity and hypophysectomy for 3 wk did not obviously affect the appearance of tight junctions.  Feeding of the goitrogen, thiouracil, which stimulates secretion of thyroid-stimulating hormone, resulted in the appearance of some very narrow and some very wide, tight junctions or sometimes junctions with both wide and narrow regions within the same cell.


Atypical gap junctions in the ciliary epithelium of the albino rabbit eye.

Gap junctions are present between nonpigmented and pigmented epithelial cells layers, as well as between the adjacent pigmented cells, but not between the nonpigmented epithelial cells which face the posterior chamber.  The unusual feature of these gap junctions is a dark banding which regularly appears every three rows of subunits.  This dark band is the equivalent width of two rows of facets.


Growth of the nuclear envelope in the vegetative phase of the green alga Acetabularia.  Evidence for assembly from membrane components synthesized in the cytoplasm.

The primary nucleus of the green alga Acetabularia grows about 25,000-fold in volume while it is separated from the endoplasmic reticulum and the whole cytoplasm by a special paranuclear cisterna of a vacuolar labyrinthum system which shows only very few (two to six per square micrometer) and small (ca. 40-120 nm in diamter) fenestrations.  The nuclear envelope does not bear polyribosomes, nor do they occur in the entire zone intermediate between the nuclear envelope and the paranuclear cisterna.  It is suggested that this special form of nuclear envelope growth takes place by assembly from cytoplasmically synthesized proteins that are translocated across the paranuclear cisterna in a nonmembrane-structured form.


Cell surface lipids and adhesion. I. The effects of lysophosphatidyl compounds, phospholipase A2 and aggregation-inhibiting protein.

Aggregation-inhibiting protein (AIP: Curtis & Greaves, 1965), which diminishes the adhesiveness of cells, particularly at low temperatures, is identified in the present paper as phospholipase A2 (EC. 3.1.1.4). Our reasons for this identification are because phospholipase activity parallels AIP activity on cell adhesion, and because various inhibitors and sera act in a parallel manner on adhesion in the presence of AIP or phospholipase. We suggest that the enzyme acts on adhesion by producing lysolecithin and other lysolipids in the plasmalemma. Addition of lysolipids diminishes cell adhesion in a manner similar to phospholipase A. Incubation of cells in Hanks' medium at 37 degrees C has a parallel effect. Conditions which would be expected to stimulate reacylation of lysolipids in the plasmalemma, i.e. incubation of cells in the external presence of CoA, ATP and a fatty acid, lead to a recovery or maintenance of adhesion after or during Hanks' incubation at 37 degrees C. All these results suggest that lipid components of the cell, probably in the plasmalemma, are of importance in adhesion. The results are discussed in relation to the long-standing controversy about the effects of low temperatures and trypsinization on cell adhesion, for phospholipase treatment of cells affects adhesion in a manner similar to trypsinization.


Cell surface lipids and adhesion. III. The effects on cell adhesion of changes in plasmalemmal lipids.

The two preceding papers of this series suggest that the state of the plasmalemmal lipids affects cell adhesion. Plasmalemmal composition was altered by the experimental incorporation of fatty acids into R1 and R2 positions in the phosphatidyl components of the cell surface. In this paper we report that: (1) If the incorporation is of long chain length fatty acids (saturated) cell adhesion rises. (2) If the incorporation is of unsaturated fatty acids cell adhesion falls as the unsaturation increases. (3) Incorporation has to be extensive to produce a large change in adhesion. (4) Changes in adhesion parallel the plasmalemmal incorporation but do not follow the total cell incorporation. Item (4) argues that it is plasmalemmal and not other membrane lipids that are involved in cell adhesion. Item (3) suggests that bulk membrane properties and not some very specific grouping are involved in the effects of lipids on adhesion. The similar extents of incorporation of the various different fatty acids and the negligible amounts of lysophospholipids in the membranes of cells that have incorporated fatty acids argue that the effects are not due to differential accumulations of these lysolipids when incubations are done with different fatty acids. The changes in adhesion cannot be accounted for by changes in surface charge density since the electrophoretic mobility of the cells is unchanged by these incubations. It is suggested that these effects on adhesion due to changes in plasmalemmal lipids can be explained either in terms of the action of intermembrane van der Waals--London (electrodynamic) forces in cell adhesion or of changes in surface fluidity. These alternatives are discussed.


Mode of action of amphotericin B on chick embryo fibroblasts and on mouse Ehrlich tumour cells: a cytological and cytochemical analysis.

Chick embryo fibroblasts cultivated in vitro and Ehrlich ascites tumor cells (in vivo or in vitro) have been treated with amphotericin B. Cell multiplication is strongly inhibited. Large clear zones appear in the fibroblast nucleoi (phase-contrast and electron-microscope observations). Many treated fibroblasts and tumour cells have a high DNA content (pre-mitotic or polyploid level; measurements by cytophotometry). However, the RNA content (cytophotometry) and the total protein content (cytophotometry and micro-interferometry) are relatively low in the tumour cells. As shown by autoradiography, DNA synthesis is active but RNA synthesis and, in some cases, protein synthesis are inhibited. Due to this unbalanced growth, the cells cannot divide.


Transformation of plastids in the leaves of Acer negundo L. var. odessanum (H. Rothe).

The leaves of Acer negundo L. var. odessanum (H. Rothe), if permanently exposed to strong sunlight, do not green, but remain yellow and finally become bleached. In yellow leaves the plastids contain single thylakoids and no grana. In plastids of bleached leaves, however, only vesicles are present. The concentration of chlorophylls and photosynthetic activity are much lower in those leaves than in the green ones. If the illumination is reduced (e.g. by shading) both the yellow and the bleached leaves become greenish, and even fully green after a few days at a sufficiently low light intensity. The plastids of yellow-green leaves contain small grana. In dark green leaves the thylakoid system of the chloroplasts is normally developed forming true grana, regardless of whether the leaves were originally green, or became green by shading the yellow or bleached ones. Their pigment concentration and photosynthetic activity are also normal. If green leaves are exposed to sunlight they do not yellow or bleach. During a 3-week period the structure of the thylakoid system did not perceivably change, with the exception that large plastoglobules formed in  the stroma.


Hepatocyte function in long-term organ culture of Amphimuma means liver.

Fragments of liver from the adult urodele Amphiuma means, the Congo eel, were maintained in organ culture for up to 70 days. The normal electrophoretic patterns of several enzymes were retained. The activities of ornithine transcarbamylase, arginase, glutamate oxalacetate transaminase and glutamate pyruvate transaminase, and urea production, glucose uptake and tissue glycogen content remained relatively constant throughout the culture period. Histological organization and hepatocyte ultrastructure were also retained. Liver fragments survived better in media based on MEM or BME than in medium based on Leibovitz L15. Since many aspects of tissue-specific structure and function are retained, long-term amphibian organ culture is well suited to studies on the control of hepatocyte function and on the effects of metabolites, hormones, drugs and toxins.


A new occludens-like junction linking endothelial cells of small capillaries (probably venules) of rat jejunum.

Freeze-cleave replicas of small capillaries of rat jejunum have revealed the presence of a new type of junction linking endothelial cells. This new junction reveals tight junctions (zonulae occludentes) in that the adjacent plasma membranes are held closely together along lines of attachment organized in the form of a loose, but frequently discontinuous network. In contrast to tight junctions, the A-face ridges possess a very low profile, and only at low shadowing angles can a repeating, particulate substructure occasionally be resolved. The shallow B-face furrows lack any particulate components. Images of cross-fractured focal points of attachment suggest that the external leaflets of adjacent membranes are closely apposed but not actually fused, as is the case with zonulae occludentes. It appears that this new type of endothelial junction is characteristic of small venules. Thus we propose that it be termed small venule endothelial junction.


Planning for effective use of on-line systems.

A perspective of developments in the field of computerized information retrieval is presented, and policies are suggested for the efficient use of on-line systems. Examples of MEDLINE, CHEMCON, AND TOXLINE searches are given with cost data and compared, where possible, with manual searches. The lowest costs were obtained with on-line searching by information scientists. MEDLINE is superior in quality to manual searching of Index Medicus, but on-line searching of Chemical Abstracts keywords is not an effective substitute for manual searching of Chemical Abstracts Substance Indexes.


Thin-layer gel chromatography of dyed proteins.

Plasma chromatography as a method for ultratrace qualitative and quantitative detection of organic compounds is especially well suited for detection of gas chromatographic effluents. The optimum range of sample quantity is 10(-6) to 10(-12) g for detection and identification of a compound by use of its characteristic positive and negative mobility spectra. The type of reference mobility spectra produced by alkanes, aromatics, esters, halogenated compounds, nitrogenated compounds and organic acids have been previously reported. This study presents the reference mobility spectra produced for lysergic acid diethylamide (LSD), delta9-tetrahydrocannabinol (delta9-THC), digitoxigenin and several biochemical compounds of research significance. LSD and delta9-THC in a mixture can be detected and identified by plasma chromatography positive mobility spectra in quantities of 10(-7) g or less. All the compounds investigated in this study display strong MH+ ions along with other ions primarily of the type (M)NO+, (M)2H+. None of these compounds exhibits negative mobility spectra.


Detection of lysergic acid diethylamide, delta-9-tetrahydrocannabinol and related compounds by plasma chromatography.

Plasma chromatography as a method for ultratrace qualitative and quantitative detection of organic compounds is especially well suited for detection of gas chromatographic effluents. The optimum range of sample quantity is 10-6 to 10-12 g for detection and identification of a compound by use of its characteristic positive and negative mobility spectra. The type of reference mobility spectra produced by alkanes, aromatics, esters, halogenated compounds, nitrogenated compounds and organic acids have been previously reported. This study presents the reference mobility spectra produced for lysergic acid diethylamide (LSD), delta-9-tetrahydrocannabinol (delta-9-THC), digitoxigenin and several biochemical compounds of research significance. LSD and delat-9-THC in a mixture can be detected and identified by plasma chromatography positive mobility spectra in quantities of 10-7 g or less. All the compounds investigated in this study display strong MH-+ ions along with other ions primarily of the type (M)NO-+, (M)2H-+. None of these compounds exhibits negative mobility spectra.


A simple gas-liquid chromatographic method with electron capture detection for the determination of progesterone in the blood of humans and domestic animals.

A simple routine method is reported for the determination of progesterone in blood by gas-liquid chromatography. An unlabelled internal standard, testosterone acetate, was added to plasma samples. Following preliminary purification by thin-layer chromatography, progesterone in pregnancy plasma was evaluated as the free steroid with a flame ionization detector. The hormone in the plasma of women during the menstrual cycle and of cycling domestic animals was determined as the 3-enol ester heptafluorobutyrate by electron capture detection. The validity of testosterone acetate as an internal standard was proved by simultaneous processing of [7alpha-3H]progesterone and a [4-14C]testosterone acetate and the determination of the isotope ratio in samples. The results of control experiments and normal values are also presented.


Gel chromatography of steroid oestrogens on sephadex LH-20.

The behavior of 23 steroid oestrogens Sephadex LH-20 using six solvent systems was investigated. The fractions eluted by gel column chromatography were analysed by thin-layer chromatography and spectrometry. The method was used for the radiochemical purity of labelled compounds by isotopic dilution.


Isolation and separation of tall fescue and ryegrass alkaloids.

Bases were extractable from dried, powdered grass material by percolation with methanol. The bases were removed from the methanol by cation-exchange resins and subsequently removed from the resins. Isolation of bases by these methods eliminated the problems associated with the use of large quantities of chlorinated solvents and formation of emulsions found in previously described isolation methods. The bases were separated by paper and thin-layer chromatography (TLC). TLC and a spectrodensitometer were used to quantify the principal alkaloids in crude extracts of tall fescue samples. This procedure recovered all of the alkaloids in spiked samples, and the standard deviation for perlolidine and perloline in tall fescue samples was 3% or less of the mean value.


Quantitative gas chromatographic determination of nitrilotriacetic acid in the presence of other carboxylic acids.

Nitrilotriacetic acid was separated (as its tri-n-butyl ester) from twenty-four other fatty, phenolic and polycarboxylic acids, using prepacked commercial OV-210 columns. Quantitative analysis down to 0.010 mg nitrilotriacetic acid per liter was demonstrated. The molar responses and retention times of the acids, relative to phthalic acid, were determined.


Gas chromatographic determination of micro-amounts of cyclamates.

It was found that triethylammonium cyclamate is converted into N-hepta-fluorobutyrylcyclohexylamine in a high and constant yield by reaction with hepta-fluorobutyric anhydride at 90 degrees for 1 h, and gas chromatography of the product gives a sharp peak that is highly sensitive to an electron capture detector. A useful method for the micro-determination of cyclamates was established by combining this reaction with gas chromatography.


Quantitative assay of diclofenac in biological material by gas-liquid chromatography.

Diclofenac and an internal standard are isolated from biological samples by acid-specific extraction and converted into the corresponding indolones with 0.5% sulphuric acid in 2,2,2-trifluorethanol. The neutral derivatives are isolated by solvent extraction and subjected to gas-liquid chromatography. The use of an electron capture detector permits the assay of diclofenac at levels down to 2 ng per sample.


[Determination of chloroquine in biological material by gas-liquid chromatography (author's transl)].

A method is developed for the gas chromatographic determination of chloroquine after extraction from biological material, using medazepam as the internal standard and a column filled with 3% OV-17 on Gas-Chrom Q. The detection limits are: 0.15 mug/ml of urine, 0.25 mug/ml of whole blood, and 1.50 mug/g of tissue. The technique can be applied in toxicological analysis and in monitoring drug levels.


A gas chromatographic method for measuring 6-mercaptopurine in serum.

A method is presented for the measurement of 6-mercaptopurine (6-MP) in serum. Serum samples containing the drug were treated with dithioerythritol and saturated with ammonium carbonate. 6-MP was then extracted from the serum with a mixture of isopropanol-ethyl acetate (1:1, v/v) containing 0.01% ethanethiol. The solvent was evaporated, the residue dissolved in dilute hydrocholoric acid, washed with chloroform, and extracted with ethyl acetate. 6-MP was derivatized with trimethylanilinium hydroxide and measured gas chromatographically. The sulfhydryl-protecting reagents, dithioerythritol and ethanethiol, were added to prevent the decomposition of 6-MP. The extraction and clean-up method recovered 78 +/- 2% (S.E.) of the 6-MP present. Tracer amounts of [8-14C]6-MP served as the internal standard during the extraction part of the method. Theophyline was used as the internal standard during the gas chromatographic analysis. The standard curve obtained from the gas chromatograph was linear between 0.5 and 20 mug/ml of 6-MP. Serum samples were stored in the freezer for two weeks without significant loss of drug. No interference was encountered from normal serum constituents or xanthines, such as caffeine or theophylline added to serum.


Gas chromatographic-mass fragmentographic determation of "steady-state" plasma levels of imipramine and desipramine in chronically treated patients.

A sensitive and specific method for the simultaneous determination of imipramine and desipramine in the blood plasma of depressed patients under treatment that involves separation by gas chromatography and detection by mass fragmentography is described. Concentrations were determined by focusing the mass spectrometer upon the ions at m/e 280 and 235 for imipramine and 308, 236 and 114 for desipramine (N-acetyl derivative), while promazine was used as the internal standard (ions at m/e 284 and 238). Determinations are possible at levels as low as 10 ng/ml in plasma.


Gas chromatographic confirmation of amino acid structure via diastereomer preparation.

The partial gas chromatographic (GC) resolution of several amino acid N-trifluoroacetyl 2-butyl ester diastereomeric derivatives on a short packed column has been used to confirm GC peak assignments in analysis of a marine sediment sample.


Electron-capture gas chromatography of methadone after oxidation to benzophenone.

A procedure for the determination of low levels of methadone (6-dimethylamino-4,4-diphenylheptanone-3) in serum has been developed. Methadone is extracted from serum into n-heptane and re-extracted into an acidic aqueous phase. Methadone is oxidized to benzopheone with barium peroxide in sulphuric acid, during which procedure an n-heptane phase is present into which the oxidation product is continuously extracted. The benzophenone formed is determined by means of electron-capture gas chromatography. The recoveries are 100 +/- 3% and 100 +/- 4.5% at the 120 and 16 ng levels, respectively. The minimum amount that can be determined in 1 ml of serum is 4 ng. Interferences from possible metabolies are probably minor. The main cyclic metabolite is only co-determined to a minor extent if the oxidation time is optimized. Comparison of this oxidation method with a combined gas chromatographic-mass spectrometric determination with selected ion monitoring showed identical serum levels.


Gas chromatographic determination and gas chromatographic-mass sepectrometric analysis of chloramphenicol, thiamphenicol and their metabolites.

A rapid and accurate gas chromatographic (GC) method is described for the determination of chloramphenicol, thiamphenicol and their metabolites excreted in human urine. These excretions were pre-treated with diazomethane and N,O-bis(trimethylsilyl)acetamide, so that they could be easily subjected to GC with satisfactory separation from each other and also from other urinary excretions, and could be determined simultaneously. The structures of the metabolites were confirmed by GC combined with mass spectrometric measurements of the GC peaks. The application of the method to urine specimens enabled more precise results for the amounts of metabolites present to be obtained; the excretion of thiamphenicol glucuronide was not observed.


Combined ultraviolet-fluorescence detection in high-pressure liquid chromatography of pharmaceuticals.

The use of combined UV-fluorescence detection for the evaluation of incompletely resolved compounds and trace components in the presence of large quantities of major components is described, analysis for thioridazine and some of its oxidation products by high-pressure liquid chromatography being chosen as a practical example. Mesoridazine and the ring oxide of thioridazine have been determined quantitatively with relative standard deviations (n = 6) of 2.0 and 3.6%, respectively, at concentrations below 0.1 mug per injection. Resolution of the two components is difficult and, in this instance, unnecessary. By a similar approach, it was possible to determine the highly fluoresecent sulforidazine at a concentration of 0.4% of the thioridazine with 6.2 mug of thioridazine injected. A relative standard deviation of 5% was attainable at this concentration. Fluorescence detection limits for mesoridazine and sulforidazine at a signal-to-noise ratio of 4:1 are between 5 and 10 ng per injection; this corresponds to about 0.1% of the active substance for the above example.


Agarderivatives for chromatography, electrophoresis and gel-bound enzymes. IV. Benzylated dibromopropanol cross-linked sepharose as an amphophilic gel for hydrophobic salting-out chromatography of enzymes with special emphasis on denaturing risks.

The preparation of benzylated covalently cross-linked Sepharose 2B is described. Such gel was analyzed for its degree of substitution, and gels with three different degrees of substitution were used in chromatographic experiments with dextranase, alpha-amylase, lactate dehydrogenase, alpha-chymotrypsin and trypsin. Yields and chromatographic patterns for different eluting systems were determined. It was found that gradients combining an increase in ethylene glycol concentration with a decrease in salt concentration gave better results than did pure salt gradients. No denaturation was observed for dextranase or alpha-amylase, but the other enzymes tested were partly denatured. The most severe denaturation was observed for lactate dehydrogenase desorbed from the highest substituted gels, although the enzyme was highly active in the adsorbed state. The results and the use of amphophilic gels are discussed.


Determination of nitrazepam in serum by gas-liquid chromatography. Application in bioavailability studies.

A gas chromatographic method with electron capture detection has been developed for the analysis of nitrazepam in serum. N-Desmethyldiazepam is used as internal standard. Nitrazepam isolated from serum is converted by acid hydrolysis into 2-amino 5 nitrobenzophenone, which is chromatographed. Metabolites of nitrazepam (7-amino and 7-acetamido compounds) are not included in the determination. Recovery experiments showed that the method is quantitative. The limit of detection is 5 ng/ml of nitrazepam in serum. The method has been used for measuring serum concentrations of nitrazepam in bioavailability studies on subjects given a single dose of nitrazepam tablets.


Controlled pore glass chromatography of protein-sodium dodecyl sulphate complexes.

The inclusion of urea has been found to eliminate adsorption of protein-sodium dodecyl sulphate (SDS) complexes to controlled pore glass. Using buffer containing 6 M urea, 0.5% SDS and glass with pore diameter 12.3 nm, it is possible to determine protein molecular weights in the range 3500-12,000. Results with glass of larger pore diameter (25.5 nm) are similar to those reported in the absence of urea in the molecular-weight range 12,000-140,000. Controlled pore glass chromatography also permits the study of the relative importance of conformation free of charge effects for those proteins which deviate from the normal calibration curve for SDS-polyacrylamide gels.


Separation of the methylated nucleoside consituents of ribonucleic acid by two-dimensional thin-layer chromatography.

In an attempt to separate a number of methylated nucleoside constituents reportedly present in various mammalian RNA preparations, a comparison of the migration of selected nucleoside standards was assessed via thin-layer chromatographic techniques with respect to three solvent systems and an adsorbent of varying proportions of silica gel and microcrystalline cellulose. The results of these studies revealed that a mixture of seventeen methylated constituents could be separated following two-dimensional development on an adsorbent containing 60% cellulose and 40% silica gel. Constituents separated included the 2'-O-methylated derivatives of adenosine, guanosine, cytidine and uridine, 1-, N6- and N6,N6-methyladenosines, 1-methylinosine, 1-, 7-, N2- and N2,N2-methylguanosines, 3-, 4- and 5-methylcytidines and 3- and 5-methyluridines. In addition, all of the above constituents were separated from their respective non-methylated nucleosides and bases.


The direct gas chromatographic analysis of barbiturates as their sodium salts.

The addition of formic acid to the carrier gas will permit the on-column liberation of the free acids following the direct injection of barbiturates as their sodium salts. These free acids may then be resolved upon an Apiezon L column: the presence of formic acid also minimizes the adsorption effects which may be associated with the gas chromatographic analysis of these relatively polar drugs.


Rapid analysis of individual tablets by high pressure reverse phase liquid chromatography.

A high pressure liquid chromatographic determination of a steroidal oxime in the pharmaceutical dosage form is reported. The reversed phase liquid-liquid partition chromatography employing ODS-Permaphase as support allows thirty to forty individual tablets to be analyzed per hour at a dose level of 50 micrograms to 1 milligram per tablet. The method is simple, rapid, accurate and sensitive.


Gas chromatographic evaluation of the volatile constituents of lung, brain and liver tissues.

Volatile metabolites from rat liver, lung and brain tissues were compared using gas chromatography. Volatiles released from the homogenized tissues at 95-100 degrees C were collected on a poly phenyl ether solid adsorbent. The adsorbed volatiles were examined by high-resolution gas chromatography. Markedly differing overall volatile profiles were observed for the tissue types examined, and it appears that certain constituents may be characteristic of a particular tissue.


The examination of some vinyl acetate/olefin copolymers by pyrolysis gas chromatography mass spectrometry.

A study of the thermal degradation of some vinyl acetate/olefin copolymers reveals that side chain scissions at the backbone and scissions within the side chains themselves yield fragments which are apparently characteristic of the parent olefin.