The carbohydrate structure of a glycopeptide released by the action of plasma kallikrein on bovine plasma high-molecular-weight kininogen. Fragment 1, released from bovine plasma high-molecular-weight kininogen by the action of plasma kallikrein, is a glycopeptide with approximately 3 mol of each of N-acetylgalactosamine, galactose and sialic acid in one molecule. All these sugars were in the T-1 fragment obtained by tryptic digestion of fragment 1. Sialic acid can be completely released from the T-1 fragment by sialidase digestion. When this sialic acid-free T-1 fragment was incubated with purified diplococcal endo-alpha-N-acetylgalactosaminidase, all remaining sugars were released as a disaccharide. By Smith degradation and beta-galactosidase digestion, the structure of this disaccharide was found to be Gal beta 1 leads to 3GalNAc. Methylation analysis of the trisaccharide released from fragment T-1 by alkaline borohydride treatment indicated that all the galactose was obtained as the 2,4,6-tri-O-methyl derivative. Based on this evidence, the complete structure of the carbohydrate moieties of fragment 1 was proposed as Sialyl alpha 2 leads to 3Gal beta 1 leads to 3GalNAc. In addition, small amounts of a tetrasaccharide, Sialyl alpha 2 leads to 3Gal beta 1 leads to 3(Sialyl alpha 2 leads to 6)GalNAc also occurred as a carbohydrate chain of fragment 1.


Studies of peroxisomes. VIII. Evidence for framework protein of the cores of rat liver peroxisomes. The cores of peroxisomes were purified 670 fold from a rat liver homogenate and the protein in the preparation was examined by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. Two bands of protein were detected on 10% polyacrylamide gel, and their molecular weights were calculated to be about 32,000 and 27,000. On treatment of the core fraction with alkali, urate oxidase was solubilized and on 10% polyacrylamide gel this fraction gave a single band of protein with an estimated molecular weight of 32,000. These results suggests that the protein component having a molecular weight of 27,000 is the framework protein of the core of rat liver peroxisomes.


Studies on papain catalysis with substrates containing the trans-p-phenylazobenzoyl group as a probe for hydrophobic interaction. The susceptible bonds of substrates of the type PABz-GLYn-Arg-R (R=OCH3, n=0,1,2; R=OH, n=1,2) to papain [EC 3.4.22.2] have been identified. PABz-Arg-OMe was found to  be approximately 30 times more reactive than benzoylarginine ethyl ester, due to a Km(app) value 25 times smaller. The finding that PABz-Gly2-Arg-OMe was specifically hydrolyzed at the ester bond with a second-order rate constant comparable with that of PABz-Arg-OMe indicated that the S4 subsite of the enzyme is capable of accommodating a large hydrophobic group. Other substrates were cleaved at the peptide bond one residue removed from the PABz group, in accord with the well-known fact that the S2 subsite interacts preferentially with a hydrophobic P2 residue. A negative charge on the substrate at the P2' or P3' position greatly decrease the acylation rate and also the noncovalent binding affinity. The hydrolysis of PABz-Arg-OMe and PABz-Gly2-Arg-OMe was little affected by the acridine dye proflavine.


Isolation of nonhistone-protein-rich chromatin fragments differing in composition and recovery between transcriptionally active and inactive sources. Soluble chromatin was fragmented by deoxyribonuclease I[EC 3.1.4.5] or II[EC 3.1.4.6] digestion or sonication, followed by 0.7 m NaCl treatment, which preferentially removed DNA fragments and histones from the fragmented chromatin, but not from intact chromatin, leaving an insoluble fraction of DNA-histone-nonhistone protein complex  without appreciable redistribution of the chromatin components. Fragmentation of DNA and concentration of nonhistone proteins in the fraction were most effective with deoxyribonuclease I under the conditions used. Thus, only 1% of the DNA in transcriptionally inactive chicken erythrocyte chromatin was recovered, together with 2% of the total histone and 50-100% of the total nonhistone protein. Separate experiments showed that this DNA fraction was among the double-stranded segments having in average size of 210 base pairs and was associated with histone components responsible for the nucleosome conformation as well as most of the nonhistone protein components in the original chromatin. From transcriptionally active  chicken liver or calf thymus chromatin, however, 5-10% of the total DNA, 20-40% of the histone, and 80-100% of the nonhistone protein were recovered  in  the corresponding fraction. These results may reflect the native states and template activities in situ of various chromatins.


Fluorometric studies on the light chains of skeletal muscle myosin. I. Effects of temperature, ionic strength, divalent metal ions, and nucleotides. Light chains of skeletal muscle myosin were studied through the reactivity of their SH groups with a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM). The experiments were carried out by reacting the reagent with myosin for a short time and measuring the amounts of reacted dye by fluorometry after separating light chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two classes of light chains, alkali light chains and DTNB light chain, were clearly distinguished by their manner of reactivity change, and differences in their environment and in their function were suggested. Although we found that the SH groups of the DTNB light chain were susceptible to very low concentrations of Mg ions (of the order of 10-5 M), we could not observe Ca2+-induced conformational change by our technique. We also estimated the stoichiometry of light chains in skeletal muscle myosin to be 1.37 mol alkali light chain 1, 1.95 mol of DTNB light chain and 0.77 mol of alkali light chain 2 per mole of myosin from the total amounts of our reagent that reacted with each light chain.


Large-scale preparation of cytosolic and mitochondrial asparatate aminotransferases from pig heart. A procedure is described for the large-scale preparation of the cytosolic and mitochondrial isoenzymes of aspartate aminotransferase from pig heart. The procedure consists of: 1. extraction of both isoenzymes by heat treatment of homogenates prepared from minced and frozen heat muscle; 2. separation of each isoenzyme on a hydroxyapatite column; 3. purification of each isoenzyme by combinations of heat treatment, ammonium sulfate fractionation and chromatography on ion-exchange cellulose columns. Purified preparations of each isoenzyme thus obtained were homogeneous proteins as judged from their spectral properties and behavior on polyacrylamide gel electrophoresis. Using the present procedure, 1.2 g of the cytosolic isoenzyme and 1.7 g of the mitochondrial isoenzyme were obtained from 20 kg of minced heart muscle.


Studies on the synthesis of proteinase inhibitors. I. Synthesis and activity of nonapeptide fragments of soybean Bowman-Birk inhibitor. Two heterodetic cyclic nonapeptides, X-Cys-Thr-Lys-Ser-Asn-Pro-Pro-Gln-Cys-Y (Ia: X = Ac,  Y = NH2; Ib: X = H, Y = OH), which correspond to residues 14-22 in the sequence of Bowman-Birk inhibitor, have been synthesized by Merrifield's solid-phase method. Inhibitory activities of Ia and Ib on tryptic hydrolysis of amide and ester substrates were examined. When Gly2-Lys-Gly3 and Tos-Arg-OMe were used as substrates, the values of I50 for the peptide Ia were calculated to  be 3.6 micron and 40 micron, respectively. When Gly2-Lys-Gly3 was used as a substrate, the value of Ki was calculated to be 1.5 micron. Ia was hydrolyzed slowly by trypsin, losing the inhibitory activity. When the Lys-Ser bond of Ia  was cleved with trypsin, the modified Ia could not be regenerated by trypsin. The linear peptide S, S'-dicarboxamidomethyl-Ia also was inactive and appeared to be a good substrate. Optical rotatory dispersion studies showed that the active fragments have characteristic  conformations  which  were lost upon modification to inactive derivatives.


Protein in the food prolongs DNA synthesis in the liver of rats after starvation. After a  period on protein-free diet and then starvation,  high protein diet induces DNA synthesis in rat liver for a longer period than diet containing an equivalent amount of amino acids. The requirement for high protein diet is limited to an early part of the prereplicative period and administration of the high protein diet later did not prolong DNA synthesis.


Purification to homogeneity of spinach nitrite reductase by ferredoxin-sepharose affinity chromatography. Assimilatory nitrite reductase was purified 1,700-fold with a yield of 22% from spinach leaves with a procedure involving ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex chromatography, gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous as shown by disc and SDS-gel electrophoresis with a specific activity (mumol NO2-reduced/min/mg protein) of 140.


Colicin E3 induced cleavage of 16s rRNA of isolated 30S ribosomal subunits. Contrary to  the previous findings we found that a specific cleavage of 16S rRNA occurred when protein A, the active component of colicin E3, was interacted with isolated 30S ribosomal subunits at high concentrations. Necessary experiments to disprove the possibility of artefacts are described.


Metabolism of thromboxane B2 in man. Identification of the major urinary metabolite. One and five-tenths milligrams of [3H8]thromboxane B2 (12.2 Ci/mol) was infused at a maximum rate of 6.4 microgram/min into a healthy male volunteer. Seventy-four per cent of the infused radioactivity was recovered in the urine within 13 h. Urinary metabolites of thromboxane B2 were isolated by reversed phase partition chromatography and high performance liquid chromatography. The major urinary metabolite was found to represent approximately 16.8% of the total radioactivity in the urine. The structure of this metabolite was shown by gas chromatography-mass spectrometry to be 2,3-dinor-thromboxane B2, the product of a single step of beta-oxidation. The rate of excretion of infused radioactivity and the relative percentage represented by 2,3-dinor-thromboxane B2 in the urine of man are very similar and the major urinary metabolite identical to that previously found by us in the non-human primate.


A role for aminoacyl-tRNA synthetases in the regulation of amino acid transport in mammalian cell lines. The regulation of neutral amino acid transport was examined utilizing two temperature-sensitive aminoacyl-tRNA synthetase mutants, by comparison of their transport activities at permissive and marginally permissive temperatures. When the temperature-sensitive leucyl-tRNA -synthetase mutant tsH1 was shifted from a normal growth temperature of 34 degrees to a marginally permissive temperature for growth of 38%, a significant enhancement in the initial rate of uptake of leucine and other L system amino acids was observed when compared with that of the parental cells (CHO-S). In contrast, the uptake of A system-specific amino acids (alanine, glycine, alpha-aminoisobutyric acid) showed no significant differences relative to the parent strain. In a similar manner, a temperature-sensitive asparaginyl-tRNA synthetase mutant RJK-4 exhibited a significant enhancement of the transport activity of A system amino acids relative to its parental strain RJK-0 when the growth temperature was shifted from 33 degrees to 39.5 degrees. Preliminary kinetic data suggest that the Vmax for transport is increased when the mutants are grown under conditions of amino acid limitation. These experiments suggest that the aminoacyl-tRNA synthetase or a related product may function in the regulation of neutral amino acid transport in Chinese hamster cells.


Tunicamycin inhibits GlcNAc-lipid formation in plants. Previous studies from this laboratory (Forsee, W. T., and Elbein, A. D. (1975) J. Biol. Chem. 250, 9283-9293; Forsee, W. T., Valkovich, G., and Elbein, A. D. (1976) Arch Biochem. Biophys. 174, 469-479) have shown that particulate extracts from cotton fibers and mung been seedlings catalyze the transfer of mannose from GDP-[14C]mannose and GlcNAc from UDP-[3H]GlcNAc into lipid-linked saccharides, Concentrations of tunicamycin of 5 microgram/ml or higher inhibit the incorporation of GlcNAc into GlcNAc-pyrophosphoryl-polyprenol but this antibiotic, even at 500 microgram/ml, had no effect on the synthesis of mannosyl-phosphoryldolichol. Tunicamycin also caused a slight inhibition in the incorporation of mannose into lipid-linked oligosaccharides. The concentration of tunicamycin necessary for inhibition was dependent on the amount of particulate enzyme in the incubations.


Binding and biologic activity of glucagon in liver cell membranes of chronically hyperglucagonemic rats. Glucagon binding by liver cell membranes was examined in rats with chronically elevated plasma levels of immunoreactive glucagon (IRG) resulting from insulin deficiency, starvation, or twice daily glucagon injections. The concentration of specific glucagon binding sites was significantly reduced in the three chronically hyperglucagonemic (IRG greater than 125 pg/ml) groups as compared with nondiabetic controls and insulin-treated diabetic control rats with only mild hyperglucagonemia. A reduction in glucagon binding sites did not occur with hyperglucagonemia of 12 h or less. Despite the reduced binding of glucagon in the three chronically hyperglucagonemic groups, the ability of glucagon to stimulate cAMP production was not reduced. It is concluded that while decreased glucagon binding occures in the forms of chronic hyperglucagonemia studied, it is not associated with a reduction in the ability of glucagon to stimulate cAMP production.


Muscarinic cholinergic receptors in rat heart. Effects of thyroidectomy. The effects of thyroid status on muscarinic cholinergic receptors in the rat myocardium were investigated. The potent muscarinic antagonist [3H]quinuclidinyl benzilate was used to identify muscarinic cholinergic receptors in rat heart particulate fractions from control, hypothyroid, and hyperthyroid rats. Thyroidectomy increased specific [3H]quinuclidinyl benzilate binding to heart particulate fractions by about 60% as compared to euthyroid rat cardiac preparations. Administration of triiodothyronine to euthyroid rats decreased specific binding to muscarinic cholinergic receptors by about 20%. Scatchard analysis revealed that the cardiac particulate fraction from thyroidectomized rats contained 134 fmol of [3H]quinuclidinyl benzilate binding sites per mg of protein, as compared with 85 fmol/mg of protein found in the heart preparation of thyroidectomized rats chrinically treated with triiodothyronine. The equilibrium dissociation constant for the interaction of receptors with quinuclidinyl benzilate was the same (1 nM) in the heart particulate fractions derived from these two groups of rats. The results of this study demonstrate that thyroid hormone can regulate the number of cardiac muscarinic cholinergic receptors. Thus, the parasympathetic nervous system may participate in the cardiovascular abnormalities in different thyroid states.


Activation of glycogen phosphorylase kinase by a calcium-activated, cyclic nucleotide-independent protein kinase system. A protein kinase, which was produced from its proenzyme occurring in rat brain upon limited proteolysis by a Ca2+-dependent protease from the same tissue (Inoue, M., Kishimoto, A., Takai, Y., and Nishizlka, Y. (1977) J. Biol. Chem. 252, 7610-7616, was capable of phosphorylating alpha and beta subunits of rabbit skeletal muscle glycogen phosphorylase kinase, resulting in a marked enhancement of the enzymatic activity. This protein kinase was entirely independent of cyclic nucleotides and differed from the catalytic subunit of cyclic AMP-dependent protein kinase. The activation of phosphorylase kinase by this active protein kinase was not inhibited by a protein inhibitor of cyclic AMP-dependent protein kinase, nor by ethylene glycol bis(beta-aminoethyl ether)N',N'-tetraacetic acid, which prevented autophosphorylation of phosphorylase kinase. The proenzyme was distinguishable from cyclic nucleotide-dependent protein kinases, since it did not bind cyclic AMP and cyclic GMP, and was inactive in the phosphorylation and activation of phosphorylase kinase both in the presence and absence of these cyclic nucleotides. Neither the protein kinase nor its proenzyme showed phosphorylase kinase activity. Available evidence indicates that the Ca2+-activated, cyclic nucleotide-independent protein kinase system as well as cyclic AMP-dependent protein kinase shows an ability to stimulate glycogen breakdown as far as tested in vitro.


Kinetics of bilirubin binding to bovine serum albumin and the effects of palmitate. The binding of bilirubin to bovine serum albumin has been determined to be a second order process, first order in each reactant. Formation of the bilirubin-albumin complex was observed as an increase in absorbance at 480 nm by stopped flow spectrophotometry. The effect of long chain fatty acid on the binding of bilirubin was investigated at palmitic acid levels of 0 to 7 mol/mol of albumin at 37 degrees and pH 7.4. The second order rate constant is 1.5 X 10(6) M-1 S-1 when the palmitate/albumin ratio is less than 5 and drops to 0.5 X 10(6) M-1 S-1 when the palmitate/albumin ratio reaches 6. The equilibrium association constant for the bilirubin-albumin complex is 5.5 X 10(6) M-1 when the palmitate/albumin ratio is less than 1, 18 X 10(6) M-1 when the palmitate/albumin ratio is between 1 and 5, and 3 X 10(6) M-1 when the palmitate/albumin ratio is greater than 5. The mechanism of the effect of palmitate varies with the total palmitate level. At low molar ratios, addition of palmitate has no effect on the association rate but decreases the dissociation rate. At high molar ratios, addition of palmitate decreases the association rate and increases the dissociation rate. It is suggested that palmitate affects the ability of the protein to undergo the conformational changes needed to accommodate bilirubin.


Rotational  relaxation of the "microviscosity" probe diphenylhexatriene in paraffin oil and egg lecithin vesicles. The rotational relaxation of the widely used "microviscosity" probe, 1,6-diphenyl-1,3,5-hexatriene, was examined by the technique of  nanosecond time-resolved fluorescence depolarization. The decays of the emission anisotropy were determined at five temperatures in the range 3-31 degrees both in a reference paraffin oil and in sonicated egg lecithin vesicles. These decays were complex in both media. Marked qualitative as well as quantitative  differences were observed in the rotational behavior of the probe in the complex bilayer medium as opposed to the homogeneous reference solvent. The results are discussed in relation to the structure of the hydrophobic bilayer membrane interior and the concept of its "microviscosity".


Reactivation of thymocyte glucocorticoid receptors in a cell-free system. The specific glucocorticoid binding capacity of the 100,000 X g supernatant from thymocytes becomes rapidly inactivated with a half-life of approximately 4 h at 0 degrees. Following this inactivation, the receptor can be reactivated by a factor (or factors) present in the high speed supernatant of L929 mouse fibroblasts. The L cell activating factor is excluded from Sephadex G-25. It can be separated from the glucocorticoid receptor of L cells by chromatography of Sephadex G-150 where it elutes with the small molecular weight material. The probable molecular weight range for the activating factor is 5,000 to 20,000. Approximately 50% of the activating activity is retained after heating at 90 degrees for 15 min.


Staphylococcal nuclease and pancreatic DNase cleave the DNA within the chromatin core particle at different sites. Staphylococcal nuclease (micrococcal nuclease) and pancreatic DNase (DNase I) were used to digest HeLa chromatin core particles which had been labeled with 32P at their 5'-DNA termini. In contrast to DNase I, which cleaves core particle DNA at 10-nucleotide intervals from the 5' termini, staphylococcal nuclease cleaves core particle DNA at different sites, both fewer in number and less regularly spaced. Thus, it is unlikely that simple physical protection of DNA is the sole mechanism whereby chromosomal proteins restrict the nucleolytic cleavage of chromatin; furthermore, it seems likely that these nucleases may recognize different geometric configurations along the chromatin core particle.


Purification and characterization of CTP: cholinephosphate cytidylytransferase from rat liver cytosol. Two forms of CTP:cholinephosphate cytidylyltransferase have been identified in the cytosol of rat liver. The L-form has a molecular weight of about 2.0 X 10(5) and low specific activity. The activity of the L-form is markedly stimulated by liposomes made from rat liver lipids. The L-form is the major species present in fresh cytosol. When the cytosol is incubated at 4 degrees for 5 days, the H-form (high molecular weight) of the enzyme is generated from the L-form. The H-form consists of multiple copies of the L-form and requires a lipid-containing fraction for optimal activity. The H-form has a wide range of molecular weights with a median value of 1.3 X 10(6). It was possible to dissociate partially the H-form of the enzyme into the L-form by incubation with 0.05% sodium dodecyl sulfate. The H-form of the enzyme could be generated by incubation of the L-form with liposomes. The L-form of the cytidylyltransferase was purified from rat liver cytosol. The enzyme gave a single band when stained for protein or assayed for activity after polyacrylamide gel electrophoresis. Both forms of the enzyme were active over a wide range of pH with an optimum at pH 7.0. The K'c (pH 6.4) was 0.80. The true Michaelis constants for the forward and reverse reactions of both forms of the enzyme are given. The H-form of the enzyme from the cytosol could be precipitated by centrifugation at 100,000 X g for 1 h.


Native form of renin in the kidney.

Native form of renin in the kidney. Renin (EC 3.4.99.19) has been observed to exist in a high molecular weight (Mr greater than 50,000) and a low molecular weight from (Mr approximately 42,000) in various tissues. Little is known concerning the origin and function of the high molecular weight form of renin and its relationship to low molecular weight renin. We have found that the high molecular weight form of renin in the kidney was converted to the low molecular weight form during the extraction process. The conversion is apparenly catalyzed by an agent(s) which requires free sulfhydryl groups since blockers of sulfhydryl groups completely suppress the conversion. This conversion could not be prevented by various specific inhibitors of serine proteases nor by the metal chelator EDTA. By the use of Na-tetrathionate it was possible to preserve the renin activity of hog kidney exclusively in the high molecular weight form. Similarly, using N-ethylmaleimide it was shown that a similar high molecular weight form of renin is the exclusive form present in rat kidney. These results suggest that the high molecular weight form of renin is the native form stored in the kidney and that it is converted by an enzyme or agent requiring sulfhydryl groups to the circulating (low molecular weight) form when it is secreted into blood. Renin activity was increased to approximately 155% of the original level upon conversion.


Vitamin K-dependent carboxylation of peptide-bound glutamate. The active species of "CO2" utilized by the membrane-bound preprothrombin carboxylase. Vitamin K participates in the post-translational carboxylation of peptide-bound glutamate to form the gamma-carboxy-glutamate residues of prothrombin. The reaction requires reduced vitamin K, bicarbonate, oxygen, and a membrane-bound carboxylase. The active species of "CO2," i.e. CO2 or HCO3-, utilized in this carboxylation was determined by the low temperature method of Filmer and Cooper ((1970) J. Theor. Biol. 29, 131-145), taking advantage of the fact that menaquinone-2, in contrast to phylloquinone, is very active at 10 degrees. Microsomes from livers of vitamin K-deficient rats, were incubated in the presence of cycloheximide, avidin, NADH, menaquinone-2, 1 mM acetazolamide (to inhibit carbonic anhydrase), and either 14CO2 or H14CO3-. At 1-min intervals aliquots were removed from the reaction mixture. gamma-Carboxyglutamate was isolated from these samples by ion exchange chromatography after alkaline hydrolysis. After 1 min the incorporation of 14CO2 into gamma-carboxyglutamate was 8 to 10 times as great as that found with H14CO3-. When the carbonic anhydrase inhibitor was omited (with or without addition of exogenous carbonic anhydrase) the two incorporation curves approximated each other at a rate near that exhibited by bicarbonate alone. Similar results were obtained in a microsomal carboxylase system solubilized with Triton X-100. It is concluded that CO2 is the active species of "CO2" initially participating in the vitamin K-dependent carboxylation of preprothrombin and that neither ATP nor biotin is required for the reaction.


Sequence of the A-protein of coliphage MS2. I. Isolation of A-protein, determination of the NH2- and COOH-terminal sequences, isolation and amino acid sequence of the tryptic peptides. The A-protein of coliphage MS2 was purified to a state of sufficient homogeneity to study its primary structure. The NH2-terminal sequence was determined for the first 8 residues. Comparison with the reported sequence of R17 protein (Weiner, A. M., Platt, T., and Weber, K. (1972) J. Biol. Chem. 247, 3242-3251) shows a difference at position 6 where alanine in R17 is replaced by threonine in MS2. The COOH-terminal sequence was shown to be -Arg-Leu-Ser-Arg, confirming the existence of UAG as the termination codon of the maturation protein (Comtreras, R., Ysebaert, M., Min Jou, W., and Fiers, W. (19731 Nature New Biol. 241, 99-101; Vandekerckhove, J., Nolf, F., and Van Montagu, M. C. (1973) Nature New Biol. 241, 102; Remaut E., and Fiers, W. (1972) J. Mol. Biol. 71, 243-261). Peptides obtained by enzymatic hydrolysis with trypsin were fractionated by a combination of gel filtration and paper electrophoresis and chromatography. Thirty-eight peptides were analyzed for amino acid composition and sequence. They provide information for 312 of the 393 residues of the A-protein polypeptide chain.


Sequence of the A-protein of coliphage MS2. II. Isolation and sequence determination of chymotryptic peptides. A total of 94 different peptides have been isolated from chymotryptic digests of underivatized (35 mg) and of reduced, carboxymethylated and citraconylated (48 mg) A-protein. Their amino acid sequences provide information for 375 residues of the total of 393 amino acids of the A-protein polypeptide chain. Abnormal specificity of chymotrypsin in the digestion of citraconylated A-protein was observed.


Rat liver glucose-6-p dehydrogenase. Dietary regulation of the rate of synthesis. The rate of synthesis of rat liver glucose-6-P dehydrogenase was determined in hepatocytes isolated from rats in three different nutritional states. In induced rats (fasted 2 days and refed a high carbohydrate diet for 4 days) synthesis of the enzyme represents 0.12% of the total soluble protein synthesized. In rats fed a standard pellet chow diet synthesis of the enzyme is reduced to 0.0067% of the total soluble protein synthesized. In rats fasted for 2 days the rate of glucose-6-P dehydrogenase synthesis is too low to be detected by our methods. In addition, there is a 27-fold difference in the amount 125I-anti-glucose-6-P dehydrogenase serum bound under saturating conditions to polysomes isolated from induced and pellet-fed rats. Both of these methods indicate that there is a large increase in the rate of glucose-6-P dehydrogenase synthesis during the dietary induction of the enzyme. Methods are described for the purification of rat liver glucose-6-P dehydrogenase to homogeneity and the preparation of an antiserum specific for the enzyme. The time course for the increase in the rate of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase synthesis in fasted-refed rats suggests that the half-life for the mRNAs coding for the synthesis of both enzymes is 3 to 6 h in the induced rat.


New stereochemical analogies between iron-sulfur electron transport proteins. Active sites of Chromatium high potential iron protein (HiPIP) and Pseudomonas Aerogenes ferredoxin can be brought into equivalent orientations by assuming that their Fe4S4Sgamma4 clusters have the effective symmetry of the non-axial molecular point group Cs. Previously undetected analogies between the two proteins emerge as a result of selecting a common orientation in this mammer. Polypeptide segments connecting Cys 46 to Cys 63 in HiPIP and Cys 18 to Cys 35 in ferredoxin are analogous in the sense that they are the same length, they connect equivalent cysteinyl sulfur atoms, and they have similar, twisted antiparalled beta conformations. Tyrosine residues 19 (HiPIP) and 2 (ferredoxin) are analogous in the sense that they interact closely with equivalent inorganic sulfur atoms. To a good approximation, interactions with the polypeptide backbone and with tyrosine side chains in the two proteins place their Fe4S4Sgamma4 moieties into diastereomeric environments, which would be expected to induce different physical and chemical behavior. Circular dichroism spectra of native and super reducible HiPIP (Cammack, R. (1973) Biochem. Biophys. Res. Commun. 54, 548-554) suggest that this relationship can help to explain the contrasting oxidoreduction properties of the two proteins.


Biochemical and immunological properties of gag genecoded structural proteins of endogenous tyep C RNA tumor viruses of diverse mammalian species. The major nonglycosylated structure proteins of mammalian type C RNA tumor viruses are synthesized in the form of a high molecular weight precursor coded for by a viral gene disignated "gag". Previous genetic analysis of a prototype virus isolated of mouse origin has led to a determination of the internal arragnement of the regions within the gag gene coding for individual structural proteins. In the present study, the biochemical properties of structural proteins of type C virus isolates of additional mammalian species were analyzed. The results obtained indicate that the biochemical properties of immunologically cross-reactive proteins have been highly conserved throughout the evolution of this group of viruses. Moreover, these findings provide a means of mapping the gag genes of a broad range of mammalian type C viruses. In view of the results obtained, a new nomenclature system for type C viral gag gene-coded translational products is proposed.


Primary structure of a high potential iron sulfur protein from a moderately halophilic denitrifying coccus. The occurrence of a specific high potential iron-sulfur protein in a halophilic, denitrifying coccus provisionally assigned to the genus Paracoccus has been confirmed through primary structure determination. This protein, known as "HiPIP", has been found previously only in photosynthetic bacteria. The sequence of this 71-residue protein is as similar to the HiPEPs from photosynthetic bacteria as they, in turn, are to one another (average number of identically matching residues is 38%). Paracoccus HiPIP is a highly acidic protein in accord with general observations on the proteins of halophilic bacteria. A possible explanation for its occurrence in Paracoccus is that gene transfer is involved.


Initiator regions from the small size class of reovirus messenger RNA protected by rabbit reticulocyte ribosomes. In order to investigate the general utility of the rabbit reticulocyte cell-free translating system for isolation of ribosome-protected initiator regions of eukaryotic mRNA, we have studied ribosome protection of 32P-labeled reovirus small mRNA species. Our studies show that rabbit reticulocyte ribosomes protect initiator regions in at least two of the four reovirus small mRNA molecules. RNA sequence analysis demonstrates that 40 S subunits protect additional sequences to the 5' side of the initiator AUG triplet.


In vitro replication of mitochondrial DNA. Elongation  of the endogenous primer sequence in D loop mitochondrial DNA by human DNA polymerase beta. Whe incubated in the presence of Mn2+ as the divalent metal activator, highly purified human DNA polymerase beta performs a selective and limited replication of KB cell closed circular mtDNA. On the basis of biochemical and electron microscopic analyses of the reaction product, we demonstrate that the polymerase specifically recognizes and elongates the 9 S primer sequence in D loop mtDNA and then proceeds to copy the displaced strand. The point at which the enzyme switches template strands is most likely that at which all negative superhelical turns have been removed and an energetically unfavorable introduction of positive superhelical turns would be required for further synthesis on the initial parental template strand. The product of the reaction is an enlarged D loop that has been converted to a duplex structure. This is the first description of the capacity of a pure eukaryotic DNA polymerase to replicate a naturally occurring, specifically initiated duplex DNA molecule. Our results suggest that this system may be particularly useful in developing an in vitro duplex circular DNA replication system with purified eukaryotic components.


Localization of the enzyme system for glycosylation of proteins via the lipid-linked pathway in rough endoplasmic reticulum. A crude preparation of microsomal membranes (postmitochondrial supernatant fraction) from the magnum portion of the hen oviduct was further subfractionated using a discontinuous sucrose gradient. Preparations of purified smooth surfaced membranes and rough endoplasmic reticulum, characterized by electron microscopy and nucleic acid content, were isolated. The enzymes involved in formation of mannose-containing glycoproteins via the lipid-linked pathway were shown to be localized in the rough endoplasmic reticulum. In contrast, a galactosyltransferase that catalyzed transfer of galactose to asialo-agalacto-orosomucoid was localized in the smooth membrane fraction. There was no evidence for the involvement of lipid intermediates in the galactosyl transfer observed in this fraction.


Activation of hepatic adenylate cyclase by guanyl nucleotides. Modeling of the transient kinetics suggests an "excited" state of GTPase is a control component of the system. A three-state model developed originally from analysis of the steady state kinetics of hepatic adenylate cyclase has been extended to account for the transient kinetics of activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). In contrast to activation by Gpp(NH)p, activation of the enzyme by GTP proceeds not only without a lag phase but is of considerably lower magnitude. These differences between Gpp(NH)p and GTP can be explained by the hypothesis that GTP is hydrolyzed at the nucleotide regulatory site(s) associated with adenylate cyclase and that GTPase activity is revealed uniquely when the enzyme system is in its state of highest adenylate cyclase activity. With this hypothesis, the characteristics of activation by GTP could be simulated. The implications of this model are discussed with respect to the actions of hormones and cholera toxin on adenylate cyclase activity.


Kinetics of avian vitellogenin messenger RNA induction. Comparison between primary and secondary response to estrogen. Following either primary or secondary stimulation of cockerels with 17beta-estradiol, vitellogenin mRNA begins to accumulate in the liver after about 30 min, reaches a maximum in 3 days, and decays thereafter with a half-life of 30 h. During primary induction, accumulation of vitellogenin mRNA begins at a low rate (50 nucleotides/s/nuclear equivalent of DNA) and after 4 h, shifts to a higher rate (340 nucleotides/s/nuclear equivalent of DNA). In contrast, during secondary induction (administration of estrogen several weeks after the primary response has ceased), accumulation of vitellogenin mRNA begins at the rate of 350 nucleotides/s/nuclear equivalent of DNA and subsequently increases by about 40%. These accumulation rates result in a maximal level of vitellogenin mRNA that is approximately 1.5 times higher during secondary stimulation than that found during primary stimulation. This difference is sufficient to explain the anamnestic response to secondary hormonal stimulation that results in higher levels of circulating vitellogenin in the plasma of the rooster.


Incorporation of a naturally occurring fluorescent fatty acid into lipids of cultured mammalian cells. A fluorescent fatty acid, cis-parinaric acid (cis-PnA), has been biosynthetically incorporated by cultured mammalian cells. The fatty acid probe is predominantly incorporated into phospholipids, with a small amount of incorporation into the neutral lipid fraction. Both phosphatidylethanolamine and phosphatidylcholine are labeled with the fluorescent probe. The temperature dependence of the fluorescence intensity of cis-PnA, either biosynthetically incorporated or added as the free fatty acid, suggests thermally induced structural reorganizations in phospholipids isolated from cultured cells enriched in palmitic acid. Results are essentially identical with both forms of the probe.


Kinetics of soluble and collagen-bound aspartate aminotransferase: diffusional effects with a two-substrate enzymatic reaction. The kinetic properties of aspartate aminotransferase covalently bound to collagen are compared to those of the free enzyme. In the bound state, the enzyme exhibits a greater affinity for glutamate, but a lower affinity for oxalacetate. In order to assess precisely the contribution of diffusional limitations on the heterogeneous enzyme kinetics, a simple modeling of diffusional effects on a two-substrate enzymatic reaction is developed. According to this quantitative analysis, diffusional limitations for oxalacetate alone account for the increased and decreased enzyme affinities toward its two substrates. Consequently, coupling of the enzyme to collagen does not significantly affect its intrinsic kinetic properties.


Lipid and polypeptide components of the crystalline yolk system from Xenopus laevis. The polypeptide and lipid components of the crystallinelipoprotein-phosphoprotein from the yolk system of Xenopus laevis are described. This lipoprotein complex contains 17% lipid of which 75% is phospholipid. The phospholipid fraction consists of mostly phosphatidylcholine and phosphatidylethanolamine. The neutral lipid fraction contains mainly triglyceride. The phosphoprotein, phosvitin, has been separated from the lipoprotein, lipovitellin, by classical methods. Three polypeptide chains can be observed in the lipoprotein and their molecular weights as determined by sodium dodecyl sulfate gel electrophoresis are 105,500, 35,500, and 32,000. Phosvitin behaves abnormally on these gels, but analysis of the results suggests Mr = approximately 16,000 to 19,000/polypeptide chain. The lipovitellin component of the yolk complex contains approximately 100 bound lipid molecules/dimer. The stoichiometry of the components of this crystalline lipoprotein system is discussed in terms of the results obtained in this study and those of other workers.


Quantitation of water-soluble acylcarnitines and carnitine acyltransferases in rat tissues. The water-soluble acylcarnitines isolated from rat heart, skeletal muscle, liver, and testis have been characterized. The following acyl residues derived from the acylcarnitine fraction were found: acetyl, propionyl, isobutyryl, butyryl, alpha-methylbutyryl, isovaleryl, tiglyl, caproyl, beta-methylcrotonyl and methacrylyl. The amounts of these acylcarnitines in heart, liver, testis and skeletal muscle from fed rats were determined. Acetylcarnitine was the most abundant acylcarnitine; however, appreciable quantities of propionyl-, isobutyryl-, isovaleryl-, and tiglyl-carnitine were found. The levels of carnitine octanyltransferse, carnitine acetyltransferase and carnitine palmityltransferase activities were determined in several tissues. In addition, carnitine isovaleryltransferase and isobutyryltransferase activities were measured in heart, skeletal muscle, liver, testis and kidney. In all instances the specific activity of isobutyryltransferase was similar to the specific activity of acetyltransferase. The results are consistent with the proposal that carnitine is involved in the catabolism of branched-chain amino acids.


Photodissociation of ligands from heme and heme proteins. Effect of temperature and organic phosphate. The effect of temperature on ligand photodissociation from protoheme and the heme proteins hemoglobin (Hb) and myoglobin (Mb) has been examined. The quantum yield of photodissociation (phi) is greater at 40 degrees than at 0 degrees; in general, larger increases are seen in the less photosensitive complexes, while phi does not change in the most photosensitive complexes. The ratio of phi at 40 degrees to phi at 0 degrees is 1.8 for HbCO, 2.3 for n-butyl isocyanide Hb, 2.7 for HbO2, and 1.3 for HbNO, with initial phi values of 0.38, 0.26, 0.028, and 0.003, respectively. This pattern of quantum yield increases is seen in protoheme as well as Hb and Mb ligand photolysis. The allosteric effector inositol hexaphosphate increases the quantum yield of lignad photolysis from hemoglobin. As with temperature, inositol hexaphosphate addition has a larger effect on complexes of low quantum yield; phi increases 1.2-fold for HbCO and 2.2-fold for HbO2 at 0 degrees. The results are discussed in terms of a model containing a photoexcited intermediate (Phillipson, P.E., Ackerson, B.J., and Wyman, J. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1550-1553).


Immunological and chemical characterization of bovine preproinsulin. Fetal bovine pancreatic poly(A)-containing RNA directs the synthesis of an insulin immunoreactive polypeptide that is larger than proinsulin, preproinsulin, in the wheat germ cell-free translation system. We have characterized this peptide in detail both immunologically and chemically and have shown that it is 2500 daltons larger than bovine proinsulin (8700 daltons), possesses both insulin and bovine C-peptide-specific antigenic determinants, and contains all the tryptic peptides found in bovine proinsulin. Preproinsulin synthesized in the wheat germ cell-free system was precipitated with approximately 4-fold greater efficiency by bovine proinsulin antiserum than by insulin antiserum. Additional evidence was obtained which indicated that the preprotein folds and undergoes correct sulfhydryl oxidation less efficiently than proinsulin, perhaps due to the presence of the hydrophobic NH2-terminal extension. Automated sequential Edman degradation of bovine preproinsulin revealed the presence of an additional NH2-terminal sequence of 23 residues, preceding the B chain segment of proinsulin. The positions of 6 of the 7 leucine residues found in the bovine preproinsulin extension were identical to those reported previously for the rat preproinsulins. This close sequence similarity between the extensions of the bovine and rat preproinsulins supports the hypothesis that these molecules fulfill similar biosynthetic functions in vivo.


Glutarate semialdehyde dehydrogenase of Pseudomonas. Purification, properties, and relation to L-lysine catabolism. The lysine-induced glutarate semialdehyde dehydrogenase of Pseudomonas was purified to electrophoretic homogeneity from a mutant strain lacking delta-aminovalerate transaminase. The properties of the enzyme, including molecular weight, amino acid composition, electrophoretic behavior, and kinetic features, distinguish it from similar dehydrogenases induced in the same cell strain by hydroxyproline or by glucarate. Enzyme induction patterns and the growth behavior of a mutant deficient in glutarate semialdehyde dehydrogenase clearly relate this enzyme to the so-called delta-aminovalerate pathway of L-lysine catabolism. Induction studies also indicate that delta-aminovalerate is a better inducer of the dehydrogenase than L-lysine. Cells of a mutant strain lacking delta-aminovalerate transaminase contained higher levels of the dehydrogenase, presumably as a result of the accumulation of delta-aminovalerate, making this mutant a useful preparative source of the enzyme. The marked reduction of lysine-inducible glutarate semialdehyde dehydrogenase in a mutant strain permitted assessment of the basal levels of hydroxyproline/glucarate-inducible ketoglutarate semialdehyde dehydrogenases not possible in wild type cells.


Effect of solutes on the cold-induced insolubility of monoclonal cryoimmunoglobulins. The effects of a variety of compounds upon the cold-induced insolubility of the IgM-K cryoglobulin McE have been examined. Cryoprecipitation was found to be inhibited by certain neutral salts, ureas, amides, tetraalkylammonium salts, long chain sodium alkyl sulfates, sugars, and a number of other agents. Cryoprecipitation was enhanced by increasing the hydrophobicity of alcohols, ureas, amides, and tetraalkylammonium salts, as well as by low concentrations of many solutes. With the exception of the alkyl sulfates, inhibition was not accompanied by detectable changes in conformation of the cryoimmunoglobulin. These inhibitions and enhancements were also associated with changes in the temperature at which cryoprecipitation was initiated as well as the temperature at which a low temperature-induced conformation change occurs in the McE protein. The effects of solutes on McE are compared to results obtained with five other (two IgG, three IgM) cryoimmunoglobulins, and it is hypothesized that electrostatic and dispersion forces are primarily responsible for the cold insolubility of monoclonal cryoimmunoglobulins.


Separation and allosteric properties of two forms of UDP-glucuronate carboxy-lyase. DEAE-cellulose chromatography of partially purified preparations of UDP-glucuronate carboxy-lyase from wheat germ results in the separation of two forms of the enzyme. Both are fully active in the absence of added DPN, have indistinguishable molecular weights (210,000), but differ in charge and kinetic properties. Both are cooperatively activated by UDP-glucuronate, however Enzyme 1 is activated at lower concentrations than Enzyme 2. At low substrate concentrations (less than or equal to 5 micron), both enzymes are activated by UDP-glucose, 2 mM concentrations of activator increasing the activity of Enzyme 1 2-fold and of Enzyme 2 2.5-fold. UDP-xylose allosterically inhibits both enzymes. At substrate concentrations equal to the apparent Km values, inhibition of Enzyme 1 is much greater than that of Enzyme 2 (83 and 28% at 0.33 mM inhibitor concentration). The data suggest that synthesis of UDP-xylose is controlled both by substrate activation and product inhibition of UDP-glucuronate carboxy-lyase. The existence of a "more active" and a "less active" species of the enzyme suggests the possibility of two interconvertible forms of the same protein and the involvement of such interconversion in further regulation of UDP-xylose biosynthesis. However it is equally possible that both represent true isoenzymes.


Structural study of circulating thymic factor: a peptide isolated from pig serum. II. Amino acid sequence. The amino acid sequence of a circulating thymic factor (FTS, facteur thymique sérique) isolated from pig serum has been established as less than Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH. This sequence was obtained by Edman analysis both on the intact peptide and after tryptic digestion. The COOH-terminal residue was identified by carboxypeptidase A digestion. A synthetic peptide was prepared which showed the same biological activities as native pig circulating thymic factor, thus confirming the proposed primary structure.


Reiteration frequency of the protamine genes in rainbow trout (Salmo gairdnerii). Protamine mRNA was isolated in a very pure form from trout testis and used as a template for the synthesis of a complementary DNA of high specific activity. The cDNA represented a full copy of the mRNA template and was used in hybridization reactions with purified trout DNA to determine the number of genes for protamines in trout testis. Our results indicate that there are less than four genes for each protamine polypeptide per the content of DNA in the male gamete and that the control of protamine synthesis in trout testis cells does not involve specific amplification of the protamine genes.


Isolation and characterization of trypsin inhibitor from opaque-2 corn seeds. Trypsin inhibitor was isolated from seeds of opaque-2 corn by affinity chromatography on a trypsin/Sepharose column. The two major forms of inhibitor eluted from the affinity column were separated by DEAE-cellulose chromatography in the presence of urea. One form of inhibitor is a single-chain protein that has a molecular weight of approximately 12,500; the second inhibitor has two polypeptide chains and appears to have been produced from the single-chain inhibitor by exposure to trypsin in the affinity chromatography step. The relationship of the inhibitor isolated from opaque-2 corn to an inhibitor previously isolated from an unspecified strain of maize by Hochstrasser et al. (Hochstrasser, K., Muss, M., and Werle, E. (1967) Z. Physiol. Chem. 348, 1337-1340) is discussed.


Mouse liver metallothioneins. Complete amino acid sequence of metallothionein-I. The amino acid sequence of thionein-I, a major mouse liver thionein, is presented. Thionein-I is a single chain polypeptide which consists of 61 amino acid residues and has the amino acid composition Cys20, Asp3, Asn1, Thr5, Ser9, Gln1, Pro2, Gly5, Ala5, Val2, Met1, Lys7, with N-acetylmethionine and alanine as NH2- and COOH-terminal amino acids, respectively. Automated sequence analysis of a major cyanogen bromide peptide consisting of 60 amino acid residues, as well as characterization of the peptides obtained from trypsin and papain digestion, led to the elucidation of the complete primary structure of this protein. Remarkable structural homology is observed between the mouse thionein-I and the equine renal thionein-1B (Kojima, Y., Berger, C., Vallee, B.L., and Kägi, J.H.R. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3413-3417).


U-G-A suppressor of bacteriophage T4 associated with arginine transfer RNA. A U-G-A suppressor of bacteriophage T4, designated psu4+op, has been isolated and characterized. The transfer RNA species previously shown to have an anticodon sequence complementary to arginine codons is affected by the psu4+op mutation. Wild type and psu4+op arginine tRNAs have the same sequence except for their anticodons, where U-C-U in the wild type species is mutated to U-C-A in the psu4+op species. This mutation is believed to confer U-G-A suppressor activity on the psu4+op arginine tRNA.


Heterogeneity in nucleosome spacing.

Heterogeneity in nucleosome spacing. The organization of spacer DNA connecting 160 base-pair cores of bovine thymus polynucleosomes has been studied by a combination of biochemical and electron microscopic techniques. The results reveal that a major fraction of chromatin consists of a spectrum of repeating units; these differ from each other by up to 20 base-pairs due to variation in spacer DNA length. Those polynucleosomes which have larger spacer DNA lengths are processed by micrococcal nuclease to mononucleosomes more rapidly than those organized with shorter spacers. Although a distribution of spacer DNA lengths exists, a significant proportion of spacers that are nearest neighbors share common DNA lengths. These findings imply that functional roles may be related to the manners in which spacers are organized along chromatin fibers.


In vitro translation of avian vitellogenin messenger RNA. Administration of 17beta-estradiol to roosters induced the synthesis of vitellogenin in the liver. The mRNA that specifies this protein has been purified from the livers of estrogen-treated roosters and has been shown to have a molecular weight of 2.3 X 10(6) (Deeley, R.G., Gordon, J.I., Burns, A.T.H., Mullinix, K.P., Bina-Stein, M., and Goldberger R.F. (1977) J. Biol. Chem. 252, 8310-8319). In order to rigorously establish the identity of the polypeptide specified by this mRNA, we used a staphylococcal nuclease-treated, mRNA-dependent wheat germ cell-free translation system capable of synthesizing polypeptides as large as vitellogenin (monomer Mr = 240,000). Vitellogenin mRNA directs the in vitro synthesis of a polypeptide with the following features: (a) it co-migrates with authentic vitellogenin in SDS-polyacrylamide gels; (b) it is highly enriched for serine but is not phosphorylated; (c) it is immunoprecipitated by purified, monospecific, anti-vitellogenin antibody; and (d) it has an unusual cyanogen bromide cleavage pattern characteristic of vitellogenin. The most striking characteristic of the cyanogen bromide cleavage products is an extremely large polypeptide (Mr = 90,000) that contains two phosvitins. The kinetics of incorporation of serine and methionine into vitellogenin synthesized in the wheat germ cell-free translation system indicates that the phosvitins are located near the COOH-terminal portion of the molecule.


The identification of two intra-axonally transported polypeptides resembling myosin in some respects in the rabbit visual system. Two polypeptides (M1 and M2) which co-sediment with F-actin in an ATP-reversible way have been detected in extracts of tissue from the rabbit visual system. Both polypeptides resemble skeletal muscle myosin in their ATP-sensitive co-sedimentation with actin, while they resemble the heavy chain of myosin and the lighter polypeptide of erythrocyte spectrin in their electrophoretic mobilities. (The estimated molecular weights are: MI congruent to 195,000; myosin congruent 200,000; M2 and spectrin congruent to 220,000). M1 and M2 were labeled in the cell bodies of the retinal ganglion cells with a radioactive amino acid and subsequently recovered in tissues (optic nerve, optic tract, lateral geniculate nucleus, and superior colliculus) containing segments of the retinal ganglion cell axons. The temporal sequence of labeling M1 and M2 in these tissues indicated that both polypeptides were synthesized in the cell bodies of retinal ganglion cells and subsequently transported down their axons at different maximum velocities. The estimated velocities were: M1, 4-8 mm per day; and M2, 2-4 mm per day.


Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail. The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.


Studies of the secretory process in the mammalian exocrine pancreas. I. The condensing vacuoles. Phosphatase cytochemistry was used to distinguish between the Golgi apparatus and GERL (considered as a specialized region of endoplasmic reticulum [ER] at the inner [trans] aspect of the Golgi stack) in pancreatic exocrine cells of guinea pig, rat, rabbit, and hamster. The trans element of the Golgi stack exhibits thiamine pyrophosphatase (TPPase) but no acid phosphatase (AcPase) activity. In contrast, GERL shows AcPase but no TPPase activity. The nascent secretory granules, or condensing vacuoles, are expanded cisternal portions of GERL. Continuities of condensing vacuoles with rough ER are suggested, and it is proposed that some secretory components may have direct access to the condensing vacuoles from ER. Connections of Golgi apparatus with GERL were not seen.


Localization of acetylcholine receptors in central synapses. The localization of cholinergic receptors in brain synaptosomes and in synapses of the midbrain reticular formation and hypothalamic preoptic nucleus has been demonstrated by means of a horseradish peroxidase-alpha-bungarotoxin (HRP-alpha-Btx) conjugate. Only a small proportion of the total number of synapses was reactive. Axon terminals of reactive synapses contained primarily small clear vesicles, while synapses characterized by large numbers of dense core vesicles were unreactive. Toxin-binding sites were found to occur in a thickened zone of the postsynaptic surface. This procedure can be employed to study the regional distribution and localization of nicotinic receptor sites in the central nervous system.


Isolation of pure cholinergic nerve endings from Torpedo electric organ. Evaluation of their metabolic properties. Pure cholinergic nerve endings (synaptosomes) were isolated from the electric organ of Torpedo by a rapid procedure. These synaptosomes are approximately 3 micron in diameter. They contain an occasional mitochondrion, numerous synaptic vesicles, and sometimes an active zone is observed. No postynaptic membrane attachment is found. This nerve ending fraction is extremely pure as shown by morphological controls and biochemical data. It is rich in choline acetyltransferase (450 nmol/h per mg protein) and acetylcholine (ACh) (130 nmol/mg protein). The isolated endings retain their cytoplasmic components and they synthesize ACh and are stable in vitro for several hours, as shown by biochemical measurements and morphological analysis.


[Autotransfusion in surgical practice (author's transl)]. From their experiences on animals and from its surgical applications the authors can say that the procedure of autotransfusion in vascular operations is worthwhile and safe. Elements of blood auto transfused are a little or not at all altered. Autotransfusion reduces requiremnts of bank blood, rare and containing risks.


[Hyperlipemia. Post-operative fat embolism, with psychiatric signs, and persistent lipuria (author's transl)]. The authors report an exceptional case of fat embolism with neuro-psychiatric signs, following a digestive by-pass operation for hypercholesterolemia. They emphasize the difficulty in diagnosis and attach value to the lipuria when it is massive. A short review of the literature recalls the various pathogenic theories of fat embolism.


[Surgical management of left colon and rectal radiation injuries (author's transl)]. A. First of all, we can affirm after the analysis of 132 records: the predominance of gynecologic cancers and the frequent responsibility of medical associations in the determinism of advanced radiation injuries of colon and rectum; the typically variable appearence of these injuries with an usual delay going from 6 months to a year and limits from 2 months to 35 years; the difficulty of diagnosis between radiation injurie and recurrence of cancer especially in case of fistula and the severe forecost in case of cancer radiation injurie association. B. The surgical management exist only for non-indications and failures of medical treatment; the one stage resection with end to end anastomosis will be made exclusively on advanced, therefore non evolving and limited injuries; in most cases, the multiple stage resection must be preferred: first derivation in selected part (sigmoid or transverse colon) and secondary resection in healthy area; as regards the closure of colostomy, it must never occur before a 6 months delay and anastomosis radiologic check.


[A year's practice of surgery for arteritis after the age of 70 years (author's transl)]. Surgery of the elderly arteritic raises the problem of operative risk due to pre-existing defects at an advanced stage of the arterial disease and the danger of tiring investigations, anesthetics and repeated operations. Out of 271 arteritics operated on during 1975, half (135) were aged over 70 years. 92, i.e. two thirds were operated on at the terminal stage of their arteritis. The operative indications depended on the clinical, arteriographic, psychological and sociological data. The past history in many cases of heart attacks and cerebro-vascular accidents made the utility and necessity of the operation debatable. The technical choice, sympathectomy or plastic operation, depended on which presented the less risk. Although amputation is still necessary for massive gangrene (50 p. cent survival) one should always attempt to save the leg. Lumbar sympathectomy seems less aggressive; reconstructive surgery is, with greater risk, more effective at stage IV. The final result of the year showed 72.6 p. cent survivors including 56.3 p. cent successes and 16.3 p. cent amputations and a mortality of 27.4 p. cent. But all the failures were situated at stage IV of the arteritis, and the disturbances are increased by the patients' age. We can thus say that it is possible to render survival comfortable for three quarters of our operated patients.


Determination of 8-methoxypsoralen in plasma by electron capture gas chromatography. A method is given for the determination of 8-methoxypsoralen in human plasma at the low ng/ml level using gas chromatography with electron capture detection. 8-Methoxypsoralen was extracted from plasma with methylene chloride at pH 7.0. After addition of the internal standard, 8-butoxypsoralen, the psoralens were hydrolysed in sodium hydroxide and the aqueous phase was purified by extraction with methylene chloride and toluene. The aqueous phase was acidified and the re-lactonized psoralens were extracted with toluene and analysed. Some determinations of plasma levels of 8-methoxypsoralen after oral administration are presented.


Simplified method for the determination of residues of carbofuran and its metabolites in crops using gas-liquid chromatography-mass fragmentography. The gas-liquid chromatographic behaviour of 2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate (carbofuran), its 3-keto-, and 3-hydroxy-derivatives, their respective phenolic hydrolysis products and the heptafluorobutyryl (HFB) derivatives of the carbamates and phenols were studied by examining the column effluent using chemical ionization mass spectrometry. In contrast to the behaviour of the carbamates, their HFB derivatives consistently produced ions having intensities proportional to the quantities injected. The common base-peak ion at 228 a.m.u. was used to quantitate these materials at the 0.02-1 ppm level in field-treated carrots, celery, tomatoes and corn with minimal sample preparation.


Analysis of C19O3 steroids by thin-layer and gas-liquid chromatography and mass spectrometry. The separation of twenty-six saturated and two unsaturated C19O3 steroids has been studied by thin-layer chromatography on silica gel F254, with seven mobile phases, and by gas-liquid chromatography on packed columns with four stationary phases; combination of both techniques permitted separation of all the test compounds. The mass spectra of the steroids were obtained by gas chromatography-mass spectrometry and are presented. Fragmentation processes have been studied, and characteristic ions that may be used for multiple ion detection or lead to identification of biologically produced C19O3 steroids are discussed.


Single-step separation of major and rare ribonucleosides and deoxyribonucleosides by high-performance liquid cation-exchange chromatography for the determination of the purity of nucleic acid preparations. A method is described for the separation of 13 major and minor ribo- and deoxyribonucleosides in a single chromatographic run using high-performance liquid chromatography on strongly acidic cation-exchange columns. The method proved useful for the routine determination of small amounts of ribonucleic acid impurities in deoxyribonucleic acid preparations and vice versa. About 3% or even less of nucleic acid contamination in a given sample can be easily detected and quantitatively determined under the conditions used.


High-performance thin-layer chromatographic determination of psychopharmacologic agents in blood serum. High-performance thin-layer chromatography affords a rapid, sensitive method for determination of psychopharmacologic agents in blood serum samples. Quantitation of the representative drugs chlorpromazine, amitriptyline, nortriptyline, imipramine, and desipramine at levels as low as 5 ng/ml is demonstrated by scanning the developed thin-layer plates with a chromatographic spectrophotometer in the ultraviolet absorption mode. Neither derivatization prior to, nor color development after chromatographic separation is required to achieve sensitivity and reproducibility of determinations.


Quantitation with high-performance thin-layer chromatography and programmed multiple development with high-performance micro-thin-layer material for drug analyses in biological fluids. The programmed multiple development thin-layer chromatography (PMD-TLC) technique was compared with the conventional chromatographic technique on both normal and HPTLC plates. The potency of this high-performance TLC is illustrated and discussed by means of the data obtained with the determination of digitoxin in human serum. Improvement of the efficiency of TLC, resulting in a better resolution and sensitivity with PMD-TLC using using HPTLC plates, makes this high-performance TLC technique comparable to high-performance liquid chromatography. This TLC approach might lead to a sensitive, rapid, selective and simply assay for routine serial analyses and, because of its specificity and flexibility, it may facilitate drug interaction studies.


Thin-layer densitometric determination of muzolimine (BAY g 2821), a structurally new diuretic drug, at the nanogram level in biological fluids. A sensitive and specific thin-layer densitometric method was developed for the determination of muzolimine (BAY g 2821), a structurally new diuretic drug of the pyrazolinone type. For detection of the drug on thin-layer chromatographic plates, a colour reaction with 4-dimethylaminocinnamaldehyde is carried out. The detection limit was 50 pg per spot on commercially available silica gel 60 plates. A nearly linear relationship between integrated peak area and amount per spot was obtained in the range from 100 pg to 1 mug per spot. For quantitative determination in biological fluids, a recovery of about 100% is achieved by a single extraction with dichloromethane, and the extract is spotted directly on to the plate. The detection limit of muzolimine in plasma and urine was 1 ng/ml, and inaccuracy in the nanogram range was found to be 5-8%. The assay was applied to pharmacokinetic studies and can be recommended for monitoring plasma levels of muzolimine in patients.


Gas-liquid chromatography-mass spectrometry of phospholipid mixtures after enzymic hydrolysis. Gas-liquid chromatography-mass spectrometry (GLC-MS), employing an open-tubular Silanox-type glass column, has been applied to the products of phospholipase C hydrolysis of natural and synthetic phospholipid mixtures. The materials studied were egg lysolecithin, synthetic L-alpha-l-stearoyl-2-oleoyl lecithin, bovine brain sphingomyelin, and phospholipids derived from human arterial tissue. l-Monoglycerides and ceramides were analysed as methaneboronates, and 1,2-diglycerides as trimethylsilyl ethers. The results indicate the potential value of open-tubular GLC-MS in a rapid procedure for the concurrent analysis of the major classes or polar lipids after enzymic dephosphorylation.


Organic acid profiles of human tissue biopsies by capillary gas chromatography-mass spectrometry. A method is described to determine the organic acid content of tissue specimens comparable in size to those obtainable by closed biopsy. The method involves solvent extraction of tissue homogenates followed by analysis of trimethylsilyl derivatives of the organic acids by combined capillary gas chromatography-mass spectrometry. Organic acid profiles of human liver, pancreas, kidney and muscle are shown.


Volatile substances in blood serum: profile analysis and quantitative determination. Alcohols, ketones and aldehydes have been identified in the profiles of volatile substances in blood serum. A 5-ml sample is required in order to obtain a complete gas chromatographic profile, selective profiles of alcohols or individual ketones by computer mass fragmentography, and to permit mass spectrometric identification of the compounds. The quantitative determination of total 4-heptanone using an extraction and gas chromatographic-mass fragmentographic procedure demonstrated concentrations of 10-50 nmole/l in normal serum, whereas in the serum of patients with chronic renal insufficiency the concentrations were 10- to 40-fold higher. Ethanol and total acetone were quantitatively measured by direct injection of serum. For 10 patients, concentrations between 25 and 85 mumole/l of acetone and 10-170 mumole/l of ethanol were determined.


Gas chromatographic procedure for the determination of meta- and para-hydroxyphenylacetic acids. m- and p-Hydroxyphenylacetic acids have been identified and quantitated in rat brain by gas chromatography of their pentafluoropropionyl hexafluoroisopropyl diesters. The procedure differs from those employed in earlier studies on the catecholic acids in that a support-coated open-tubular column was used, a preliminary purification of the acids by thin-layer chromatography was carried out, and excess derivatizing reagent was removed by extraction with a phosphate buffer.


Quantitation of deuterated and non-deuterated phenylalanine and tyrosine in human plasma using the selective ion monitoring method with combined gas chromatography-mass spectrometry. Application to the in vivo measurement of phenylalanine-4-monooxygenase activity. A specific method is described for the quantitative analysis of deuterated and non-deuterated phenylalanine and tyrosine in human plasma by gas chromatography-mass spectrometry using selective ion monitoring. From the several derivatives investigated, the N- or N,O-trifluoroacetyl methyl esters were found to be the most suitable for our purposes. DL-Phenylalanine-4-d1 and L-tyrosine-d7 were used as internal standards. The sensitivity of this method permits the measurement of amounts as small as ca. 2.5 ng/ml in plasma for both phenylalanine and tyrosine. The coefficients of variation were found to be ca. 1.6% (n = 12) for phenylalanine and 3.0% (n = 12) for tyrosine. Using this method, an in vivo determination of phenylalanine-4-monooxygenase activity in humans is possible by loading the subjects with deuterated L-phenylalanine-d5 (accepted as substrate by phenylalanine-4-monooxygenase E.C. 1.14.16.1) and the subsequent measuring of deuterated L-tyrosine-d4 formed and residual L-phenylalanine-d5.


Quantitative determination of drugs in biological materials by means of extractive alkylation and gas-liquid chromatography. Analytical methods utilizing extractive alkylation followed by gas-liquid chromatography are described for the quantitative determination of (neo)sulfalepsine and its metabolites, chlorthalidone and chlorquinaldol. The applications described indicate that, for the use of this technique, careful optimization and the introduction of a suitable internal standard at the beginning of the analytical manipulations are essential.


Direct gas chromatograph-mass spectrometer connection of glass capillary columns for the analysis of serotonin and metabolites by selective ion monitoring. A direct connection system that requires no modifications to either the gas chromatograph or the mass spectrometer is described for the coupling of glass capillary columns to a commercial mass spectrometer. Vacuum-tight connections can be readily achieved with standard 1/16-in. fittings and septum discs. The system can be operated with column outlet helium flow-rates of 1.5-2 ml/min at ion source pressures of 2-5 - 10(-5) mmHg. Although an injector splitter of our own design has been used, the initial test results obtained with a splitless injection method based on the use of a solids injection syringe together with a packed column injector assembly indicate the feasibility of developing a totally splitless and connection system that is simple to use. The system has been applied to the detection of the pentafluoropropionyl derivatives of serotonin, methoxytryptamine, methoxytryptophol and 5-hydroxytryptophol by selective ion monitoring techniques. Column performances under both sets of conditions (gas-liquid chromatography alone and combined with mass spectrometry) are compared and the evaluation of direct versus indirect coupling showed yields of the order of 50% through the molecular separator.


Preparation, gas chromatography and mass spectrometry of methyl and trimethylsilyl esters of indomethacin. The preparation and gas chromatographic-mass spectrometric behavior of the methyl and trimethylsilyl esters of indomethacin, 1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid, are described. Reaction of this anti-inflammatory drug with diazomethane or bis(trimethylsilyl)acetamide forms the expected esters. Derivatization with dimethylformamide dimethylacetal yields two compounds, the methyl ester (major product) and a methyl ester-dimethylaminomethylene condensation (at the alpha-carbon of the side chain) product (minor). Experiments with 5-O-desmethyl-indomethacin have demonstrated that using the described diazomethane methylation conditions no alkylation of the phenolic group occurs. Esterification combined with an isolation procedure allows the determination of indomethacin levels in plasma and aqueous humor of rabbits, the 4-fluorobenzoyl analog serving as internal standard. The derivatives exhibit excellent electron capture properties allowing quantitative assay of the drug at the submicrogram level. Precision and accuracy for plasma samples varied from 92 +/- 19% (5 ng/ml) to 96 +/- 1.5% (1000 ng/ml). The analogous values for aqueous humor are superior: 97 +/- 5.6% and 99 +/- 2.2%, resectively.


Capillary gas chromatography of s-triazines. In order to achieve better resolution and lower detection limits for s-triazines, glass capillary columns made of soft soda-lime glass and etched with gaseous hydrogen chloride have been introduced. Columns with non-polar (OV-101, SE-30) mixed (Carbowax 20M + SE-30) and polar stationary liquids (Carbowax 20M) were used. The thickness of the ultra-thin film columns was determined by weighing the capillaries before and after coating of the capillary with stationary phase and conditioning, and varied from 0.05 to 0.5 mum. From the capacity ratios of s-triazines and n-alkanes measured in sections of an originally long dynamically coated column after it had been cut into several equal parts, the constancy of the film thickness along the whole column was judged. The film thickness was not uniform and increased along the whole column. It was found that glass capillary columns with non-polar stationary phase are not very suitable for the analysis of s-triazine herbicides, as tailing occurs with some of them. Symmetrical peaks and the separation of 17 from 18 analysed s-triazines were achieved on glass capillary columns coated with Carbowax 20M.


Separation of amino acids and peptides on non-polar stationary phases by high-performance liquid chromatography. Microparticulate non-polar stationary phases, such as octadecyl-silica offer a rapid and efficient means for the separation of peptides and amino acids by high-performance liquid chromatography. Retention is attributed to hydrophobic interaction between the solutes and the hydrocarbonaceous functions covalently bound to the stationary phase surface. Consequently the species are eluted in the order of increasing hydrophobicity. Various peptide mixtures were analyzed by using gradient elution with increasing acetonitrile concentration in the eluent and monitoring the column effluent at 200 or 210 nm with an UV detector. The separation of angiotensins and enzymic digest of polypeptides illustrates the speed of the method which can be used to assay the purity of peptide hormones such as alpha-melanotropin and gramicidin or to analyze the composition of reaction mixtures involving peptides. The efficiency of the method is superior to that obtained on the conventionally used ion-exchanger columns, except for hydrophilic amino acids and peptides that are poorly retarded. Nevertheless, with a suitable ionic surfactant in the mobile phase, non-polar stationary phases can be used for the separation of these species as well.


Determination of paracetamol and its metabolites in urine by high-performance liquid chromatography using reversed-phase bonded supports. Paracetamol and its four main metabolites, the sulphate (S), glucuronide (G), cysteine (C) and mercapturic acid (M) conjugates, are readily separated in a synthetic mixture using slightly acidic aqueous alcoholic eluents (e.g. water-methanol-formic acid, 85:15:0.15, v/v/v) on either octadecyl silica (ODS silica) or octadecyl silica which has been further silanized to remove residual hydroxyl groups (ODS/TMS silica). The dependences of k' upon alcohol, acid and added salt concentrations are reported for both materials. The latter material gives the higher plate efficiencies and is much superior when applied to analysis of urines taken after therapeutic doses and overdoses of paracetamol. At least four additional metabolites are reported in overdose urines. Mass spectrometric analysis (high and low resolution) has confirmed the identity of M and identified one of the additional metabolites as methoxyparacetamol. Mass spectra of the remaining additional metabolites enable major structural features to be deduced. One of these metabolites may be associated with liver damage.


Solvent-generated ion-exchange systems with anionic surfactants for rapid separations of amino acids. The retention behaviour of amino acids in phase systems consisting of a hydrophobic solid support as the stationary phase and water-organic solvent mixtures containing a small amount of an anionic detergent as the mobile phase was investigated. Such phase systems are found to behave like conventional ion-exchange systems. The degree and order of retention of amino acids can be influenced by changing the temperature, the nature of the hydrophobic support, the pH and the nature and concentration of the anionic detergent, organic constituent and counter ion in the eluent. In many instances this solvent-generated (dynamic) ion-exchange chromatography shows a greater selectivity than conventional ion-exchange systems towards amino acids. The results obtained so far indicate that a complete separation of the 19 protein amino acids by applying solvent gradients or/and multi-column system is possible within 30 min.


Correlation of retention behaviour of steroidal pharmaceuticals in polar and bonded reversed-phase liquid column chromatography. For systematization of the correlation between the chemical structures of solutes and retention behaviour in liquid column chromatography, the retention volumes of 43 modified steroids on silica and chemically bonded reversed-phase columns were examined using various binary solvent systems. Retention parameters for the functional groups of the steroids were calculated according to Martin's additive rule. By comparing these values obtained on normal and reversed-phase columns, characteristic features of both packings with regard to solute structures and the solvent systems were elaborated.


Use of high-performance liquid chromatography to separate very small quantities of radio-labelled metabolites from biological tissue. In order to study the absorption, metabolism and tissue distribution of chemicals in cattle ticks Amblyomma variegatum, a method of separating and recovering metabolites in picogram quantities was required. A high-performance liquid chromatographic system has been investigated for this purpose, using tritium-labelled amitraz and some potential metabolites. Separation was carried out on Nucleosil 5 mum NO2 bonded phase. Interchangeable pre-columns were used to extend the range of compounds which could be separated in one run and to keep the main column free of radioactive contamination. Amitraz, 2,4-dimethylaniline and 2,4-dimethylformanilide were eluted from the column quantitatively without the addition of unlabelled carrier, down to quantities of 0.75 pg, 0.78 pg and 1.60 pg, respectively. Apparatus for automated collection of the column eluate in scintillation vials and a method for data processing are described.


Quantitative analysis of d-tubocurarine chloride in curare by column liquid chromatography. An improved quantitative analysis of d-tubocurarine chloride in the plant extract curare is presented. Gradient high-performance liquid chromatography on a hydrophobic stationary phase was found to be very suitable for the analysis of quaternary ammonium bases such as the complex mixture of curare alkaloids. Owing to the residual free silanol groups on the modified silica surface, the curare alkaloids are eluted from a reversed-phase column only if an electrolyte is added to the mobile phase. In order to optimize the separation, the effects of pH, the nature of the cation in the buffer and the concentration of the buffer of the retention of the alkaloids were investigated. Using a tetramethylammonium phosphate buffer at pH 4 in a gradient of water-methanol, undesirable retardation effects on the reversed-phase column could be suppressed sufficiently. As a result, an accurate method for the determination of d-tubocurarine chloride in curare was obtained. The coefficient of variation of this analysis is only 1.3%.


Determination of clomipramine and desmethylclomipramine in plasma by means of liquid chromatography. A method is presented for the determination of clomipramine and its major metabolite desmethylclomipramine in plasma. After extraction with n-hexane, the components are separated by high performance liquid-solid chromatography on silica gel and detected with a UV detector. The detection limits are 2 ng/ml for clomipramine and 10 ng/ml for desmethylclomipramine. Recoveries from plasma exceed 95% for both drugs. In routine analysis, 30-40 samples can be handled in one day. The practical use of the method is shown in plasma concentration-time curves after oral and intramuscular administration of clomipramine.


High-performance liquid chromatographic separation and photometric detection of phospholipids. A rapid and efficient method for the separation of (phospho)lipids by high-performance liquid chromatography using n-hexane-2-propanol-water mixtures as the solvent system is described. The lipid separation occurs on silica gel columns and the individual components are monitored directly by UV absorption at 206 nm. Of a total lipid extract from erythrocytes as well as suboesophageal ganglia of the snail Helix pomatia, a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are partly separated under these circumstances. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some instances, as is shown for phosphatidylcholines and sphingomyelins.


Environmental trace analysis of organics in water by glass capillary column chromatography and ancillary techniques. Products of ozonolysis. A method employing a polymeric sorbent has been used for analysis of volatile organic components in water. Trace level organics are sparged from water with nitrogen gas and are concentrated on Tenax GC prior to analysis with either flame ionization gas chromatography or gas chromatography-mass spectrometry. Glass capillary columns were used to obtain maximum resolution of chromatographic peaks. Specifically, the method has been applied to a qualitative and quantitative study of the products which result from ozonization of secondary treated domestic wastewater. The principal volatile products of ozonolysis are n-hexanal, n-heptanal, n-octanal and n-nonanal. Representative samples contained 0.7 ppb** of n-heptanal and less than 1 ppb of any of the volatile compounds.


Gas chromatographic-mass spectrometric determination of volatile organic compounds in an urban atmosphere. Polysorbimide and carbochrome, new sorbents with high temperature stability, have been used to concentrate trace amounts of atmospheric pollutants, and 136 C1-C13 organic compounds, including 126 hydrocarbons, 6 oxygen derivatives and 3 chlorides, were identified in air samples collected in the streets of Leningrad. One of the major features of the composition of the organic constituents of the urban air sampled is the presence of a number of CnH2n hydrocarbons (mainly olefins), and also CnH2n-2 and CnH2n-4 unsaturated hydrocarbons (including alpha-pinene and camphene). A comparison of the results with those of similar studies carried out on air samples from Zürich, Houston and Paris is of considerable interest in connection with the possible effect of geographical factors on the composition and lifetime of organic pollutants in the atmosphere.


Mass fragmentographic determination of lofepramine and its metabolites in human plasma and urine using deuterated internal standards. A sensitive and specific method for the determination of lofepramine and its matabolites, desipramine and 2-hydroxydesipramine, in human plasma and urine is described. Lofepramine, desipramine and 2-hydroxydesipramine were derivatized to ethyl p-chlorobenzoate, the bis(heptafluorobutyryl) derivative and teh N,O-bis(trifluoroacetyl) derivative, respectively, and then analysed by gas chromatogrphy--mass fragmentography. Corresponding deuterated compounds were used as internal standards. Determination was possible at levels as low as 2 ng/ml for lofepramine and desipramine and 20 ng/ml for 2-hydroxydesipramine.


Ion-pair liquid chromatography of steady-state plasma levels of chlorimipramine and demethylchlorimipramine. A method for the determination of chlorimipramine and its metabolite demethylchlorimipramine in the plasma of depressed patients during treatment is described. The method involves extraction of the parent drug, its metabolite and the internal standard from plasma, back-extraction into an acidic aqueous phase and re-extraction into a small volume of organic phase. Separation and quantitation are carried out by ion-pair partition chromatography with UV detection. Accurate determination if possible down to levels of 30 and 60 nmole per liter of plasma for chlorimipramine and the metabolite, respectively, when 1 ml of plasma is used. The coefficient of variation is 7.3% or less at different levels for chlorimipramine and demethylchlorimipramine. Plasma levels of the parent drug and the metabolite measured by this liquid chromatographic method and by a gas chromatographic procedure with electron-capture detection were in good agreement (r = 0.98). The steady-state plasma level of the metabolite was always higher than that of the parent drug in the 34 depressed patients investigated. The mean ratio between the metabolite and the parent drug was 2.7 +/- 1.1 (S.D.) Large inter-individual differences in the levels of the two compounds in patients receiving similar doses were found.


[Fluorometric determination of hydrochlorothiazide in body fluids by direct measurement of thin-layer chromatographic plates (author's transl)]. Two fluorometric methods for analysis of hydrochlorothiazide (HCT) are described utilizing direct measurement of thin-layer plates. The first method employs a modification of the Bratton-Marshall reaction and is therefore applicable to all aromatic primary amines. Following diazotation and azocoupling of the HCT hydrolysis product, a fluorescent group is added to the compound. For this purpose N-(1-naphthyl)ethylenediamine is first coupled with 4-chloro-7-nitrobenzo-2,1,3-oxadiazole. In the second method, the intrinsic fluorescence of underivatized HCT, following its extraction from plasma, urine or saliva, is used. It is shown that the sensitivity of this method is sufficient for estimating the kinetics following oral administration of 25 mg HCT.


Detection of benzoylecgonine in human urine. A thin-layer chromatography (TLC) method is described that can be used to detect benzoylecgonine (BE), a metabolite of cocaine, in human urine. It is a two-part procedure that can be integrated into a rapid screening program for drug abuse. The first part of the method utilizes two TLC solvent systems to identify a variety of drugs, including BE. The second part is specific for the cocaine metabolite and can be used as a confirmation method. The procedure is sensitive to 3-4 microgram/ml of BE in urine.


[Comparative chromatographic analyses of s-triazines (author's transl)]. The applicabilities of gas chromatography (GC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) for residue analysis of s-triazine herbicides are compared. With the conditions discussed the detection limit for the 14 s-triazines was determined as follows: 0.02-0.03 ng in GC alkali flame-ionization detection, 0.8 ng in GC flame-ionization detection, 1 ng in HPLC (UV detection), 3-5 ng in HPTLC (UV detection) and 8-13 ng in TLC (UV detection). The linear range in GC (AFID) was 10(4) whereas in the three liquid chromatographic methods it was 10(2). The reproducibility with a confidence limit of P = 95% had a coefficient of variation of +/- 5%. In routine analysis requiring a large number of separations HPTLC has advantages, because the time for a single separation is only 40 sec.


Three alkylation methods for the determination of indometacin in plasma by electron capture gas chromatography. The following alkylation methods for the determination of indometacin in plasma by electron capture gas chromatography are compared: 1, alkylation with diazopropane; 2, extractive alkylation; 3, alkylation by a solid-liquid phase transfer catalysed process. The drug in plasma at pH 4.0 is initially extracted with heptane containing 5% n-pentanol. The methyl ester of indometacin is based as internal standard. After alkylation to the propyl ester according to one of the three alkylation methods, indometacin can be determined down to 5 ng per sample by electron capture gas chromatography. The relative standard deviations (n = 10) at the 200 ng level are 5.1% for the alkylation with diazopropane, 7.5% for the extractive alkylation technique and 3.5% for the alkylation by the solid-liquid phase transfer catalysed process. The comparatively low value obtained by the last method indicates that decomposition of indometacin can be avoided under such mild conditions.


Separation of degradation products of double-labeled benzylpenicillin on a cation-exchange column. By using a cation-exchange resin and eluting with a lithium citrate buffer gradient, good separations were obtained for several degradation products of double-labeled benzylpenicillin.


Evaluation of allyldimethylsilyl ethers as steroid derivatives for gas chromatography-mass spectrometry. The preparation of allyldimethysilyl steroid ethers for the analysis of steroids by GC-MS has been studied in depth. The application of these derivatives is shown to be unsatisfactory due to competitive formation of allylsiloxane derivatives.


A sampling technique for organic components in human breath. An analytical sampling system was developed for trace organic components in breath. The sampling system was designed to maximize analyte concentrations while minimizing interference from exogenous contamination. Details of the sampling system design and its operation are presented.


An analytical approach to the quantitation of known drugs in human biological samples by HPLC. Although many HPLC methods are available in the literature only a fraction of these are applicable to the analysis of known drugs in human biological fluids. This paper presents the favoured approach of a laboratory involved in the quantitative assay of drugs in man for the subsequent study of pharmacokinetics and bioavailability.


Progesterone and 5alpha-pregnane-3,20-dione in peripheral blood of normal young women: Daily measurements throughout the menstrual cycle. The peripheral plasma concentrations of progesterone and 5alpha-pregnane-3,20-dione were measured daily throughout the ovarian cycle of five presumptively ovulatory women. The concentrations of progesterone and 5alpha-pregnane-3,20-dione during the follicular phase were 0.11 +/- 0.02 ng/ml (mean +/- SE) and 0.16 +/- 0.01 ng/ml, respectively. In four of these women the concentrations of both progesterone and 5alpha-pregnane-3,20-dione began to rise immediately after the luteinizing hormone (LH) peak and reached maximal concentrations five to eight days later (progesterone: 8.6 to 19.9 ng/ml; 5alpha-pregnane-3,20-dione: 1.4 to 2.8 ng/ml). The luteal phases in these women, measured from the day of the LH peak to the onset of the next menses, varied from 13 to 18 days. In the other woman with a late LH surge (day 27 of the cycle) the peak concentrations of progesterone and 5alpha-pregnane-3,20-dione were reached four days after the LH peak (8.9 ng/ml and 0.8 ng/ml, respectively). The luteal phase in this woman was 11 days. From the results of this study, it appears that the concentrations of 5alpha-pregnane-3,20-dione in peripheral blood during the luteal phase are proportional to the availability of progesterone as substrate for the 5alpha-reductase enzyme system.


Pubertal progression in the presence of elevated serum gonadotropins in girls with multiple endocrine deficiencies. Pubertal progression in the presence of abnormally elevated serum gonadotropins was followed over several years in two girls with multiple endocrine deficiencies and mucocutaneous candidiasis. Both had hypoparathyroidism and one had adrenal failure as well. A serum level of progesterone (8 ng/ml) consistent with ovulation was documented in the one girl who had regular monthly menses. High serum levels of FSH (30--60 mIU/ml) at times when serum estradiol was in the normal pubertal range (100--200 pg/ml) may indicate partial ovarian end-organ resistance or failure. Since premature menopause is associated with this syndrome, we postulate that we are observing puberty in girls whose ovaries are in the early stages of a destructive process which may eventually result in irreversible ovarian failure. A possible explanation could be the existence of increased resistance to gonadotropins at the ovarian level because of gonadotropin receptor antibodies or an inherent receptor defect.


25-hydroxyvitamin D and its binding protein in maternal and cord serum. Serum calcium, phosphorus, albumin, total protein, 25-hydroxyvitamin D (25OHD) and the vitamin D-binding protein (DBP) were measured in 30 cord sera and in 30 sera obtained simultaneously from their respective mothers. The maternal serum concentration of 25OHD (14.0 +/- 6.9 microgram/l, mean +/- SD) and of DBP (574 +/- 72 mg/l) were significantly higher than the respective cord serum concentration (8.0 +/- 4.4 microgram/l and 268 +/- 39 mg/l). The calculated concentration of "free 25OHD," however, was slightly but significantly higher in cord serum (0.44 +/- 0.24 ng/l) than in maternal serum (0.34 +/- 0.18 ng/l). Serum calcium and phoshporus were lower in maternal than in cord serum. A highly significant positive correlation was found between maternal and cord serum concentration of DBP (r = 0.59), total 25OHD (r = 0.79), "free 25OHD" (r = 0.86) and phosphorus (r = 0.73). These data indicate that the concentration of DBP is important for the evaluation of the placental transfer of 25OHD. Indeed, the concentration of "free 25OHD" is slightly higher in cord serum than in maternal serum, despite the maternal-to-fetal gradient of total 25OHD. The low fetal concentration of DBP is also unfavorable for the fetal storage of 25OHD during intrauterine life.


Renin reactivity, renin activity and renin concentration in patients with normal and low renin essential hypertension. Renin activity, concentration, substrate and reactivity were determined in normal subjects as well as in hypertensive subjects with suppressed and normal plasma renin activity. Renin substrate measurements were similar in all groups. Renin reactivity, a measure of circulating modifiers of the renin reaction, was significantly increased in both hypertensive groups. Reactivity was significantly greater in the normal renin hypertensive group than the low renin hypertensive group. Renin concentration was significantly suppressed in both hypertensive groups, but to a greater degree in the low renin hypertensives. These findings suggest that plasma renin concentration may be suppressed in most hypertensive subjects. Furthermore, plasma renin activity may be "normalized" in most hypertensive subjects by the effect of circulating modifiers of the renin reaction. While renin reactivity in the plasma of low-renin hypertensive subjects is accelerated to a lesser degree than that of the normal-renin hypertensives, this finding alone does not explain the low plasma renin activity.