New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.     

Binding sites for epidermal growth factor in nuclear fraction from rat liver

Binding sites for mouse epidermal growth factor (EGF) were observed in purified nuclear fraction from rat liver. The binding data yielded a curvilinear Scatchard plot which was decomposed into two binding components with different binding affinities. The binding component with higher affinity (Kd approximately 10(-10) M) represented approximately 15% of the total binding, while the high affinity binding sites were less than 5% of the total in the microsomes. The ligand-dependent autophosphorylation, one of the characteristic features of EGF receptor in the plasma membrane, was not observed at the site of the receptor (170 KDa) in the nuclear fraction. The binding characteristics for EGF fluctuated during the course of liver regeneration after partial hepatectomy; the binding capacity in the nuclear fraction increased in contrast to the decrease in the microsomes. However, the binding sites in the nuclear fraction obtained in the early period after partial hepatectomy consisted only of low affinity ones.     

Isolation and structure of a novel pyridine derivative from the Algerian newt, Pleurodeles waltlii

Since its discovery more than 20 years ago the structure of a strongly fluorescent compound called "pleurodeles blue" has remained unknown. Isolation of this pigment has been carried out by successive column chromatographies including epichlorohydrin-triethanolamine-Sephadex and phospho-Sephadex. The structural elucidation of a novel pyridone N-glycoside, 1-beta-D-glucopyranosylpyrid-2(1H)-one-6-acetic acid, is based on a detailed study of its high-resolution mass spectra, 1H and 13C-NMR spectra and its hydrolysis to yield D-glucose.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Outermost-cell-surface changes in an encapsulated strain of Staphylococcus aureus after preservation by freeze-drying.

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain of Staphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slime production. To quantify capsule and slime production, capsule antigen production and cellular characteristics such as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. After freeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-soft agar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volume index, capsule and slime production, and capsule antigen production were markedly decreased in parallel with the period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin was also compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2% honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer was used as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes were observed for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but the number of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 h of freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electron micrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state after freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

Inhibitory effect of very-long-chain monounsaturated fatty-acyl-CoAs on the elongation of long-chain fatty acid in swine cerebral microsomes

Characteristics of condensation and overall elongation of very-long-chain fatty-acyl-CoAs in swine cerebral microsomes were studied using radio high-performance liquid chromatography (RHPLC) and gas chromatography-mass spectrometry (GC-MS). The monounsaturated fatty-acyl-CoA depressed both the condensation and overall elongation activities of endogenous substrates and also of exogenous saturated fatty-acyl-CoA. The extent of the decrease of the elongation activity was dependent on the concentration and the chain length of the exogenous fatty-acyl-CoAs. The dependence of the condensation activity of monounsaturated fatty-acyl-CoA on the concentration of malonyl-CoA suggested that the non-Michaelis-Menten type kinetics was dominant for oleoyl-CoA, however, a normal kinetic pattern was obtained for endogenous palmitoyl-CoA and arachidonoyl-CoA with Km = 37 microM to malonyl-CoA. The condensation activity for icosanoyl-CoA (20:0-CoA) was inhibited by icosenoyl-CoA (20:1-CoA) in a non-competitive manner, which suggested that the condensation enzyme, or at least the active center of the enzyme for icosenoyl-CoA, was different from that for icosanoyl-CoA.     

A new class mutation of low density lipoprotein receptor with altered carbohydrate chains

In a monensin-resistant mutant (Monr-31) of Chinese hamster ovary cells, the O-linked sugar chains of the low density lipoprotein (LDL) receptor are altered, suggesting a mutation at a Golgi apparatus gene. In a compactin-resistant mutant (MF-2) of Chinese hamster V79 cells, the mature LDL receptor is apparently 5000 daltons smaller; the difference is due to altered glycosylation of O-linked sugar chains. Hybrids between MF-2 and Monr-31 still produced LDL receptor molecules with aberrant sugar chains; thus both mutants are in the same complementation group. Krieger and his colleagues (Krieger, M., Kingsley, D., Sege, R., Hobbie, L., and Kozarsky, K. (1985) Trends. Biochem. Sci. 10, 447-452) have classified Chinese hamster ovary cell mutants with altered LDL receptor structure into four groups: ldlA, ldlB, ldlC, and ldlD. Cell-cell hybrids between their ldl mutants and Monr-31 produced wild type mature LDL receptors with normal molecular sizes, suggesting that these compactin- and monensin-resistant mutants define a new class of LDL receptor mutant. Since both of our mutants are defective in internalization of LDL, we assign them as int mutants. This may imply a further etiology for hypercholesterolemia, and cases can now be examined for such a class.