Absorption of polyphenols by polyvinylpyrrolidone and polystyrene resins

Absorption of polyphenols by insoluble polyvinylpyrrolidone (PVP) and by polystyrene resins has been examined. Dowex-1 was the most efficient absorbent of polyphenols extracted from leaves of spinach, bean or tobacco. Dowex-50 and Amberlite XAD-2 were more effective than PVP for the removal of leaf polyphenols from solution. With purified polyphenols, Dowex-1 efficiently absorbed chlorogenic acid, flavonol glycosides and catechins but did not absorb condensed proanthocyanidins. PVP absorbed all classes of polyphenols examined but showed a low affinity for chlorogenic acid.     

Presence of cytidine 5'-tetraphosphate in commerical samples of cytidine 5'-triphosphate

A contaminant compound has been isolated from commercial samples of CTP by ion-exchange chromatography on a Dowex-1 column. It has been characterized as cytidine 5'-tetraphosphate from its ultraviolet spectrum, labile and total phosphate content, and periodate consumption. It is present in proportions from 0.3 to 3.9%, apparently regardless of the method of preparation, age of sample, or commericial source of CTP.     

Identification of naturally occurring calmodulin inhibitors in plants and their effects on calcium- and calmodulin-promoted protein phosphorylation

While studying the calmodulin activity in post-climacteric apples, a heat stable, dialyzable component that inhibited calmodulin-promoted phosphodiesterase activity was detected. The compound(s) that inhibited calmodulin activity did not bind to Dowex-50, H+ or Dowex-2, Cl- and was exclusively present in the neutral fraction. The inhibitors irreversibly bound to polyvinylpolypyrrolidone indicating their phenolic nature. Fractionation of the neutral fraction on a C18-microbondapak column and analysis for the inhibition of calmodulin-promoted phosphodiesterase activity showed significant inhibitory activity associated with fractions eluted 5 min, 15 min and 18 min after injection. Perdeuteriomethylation and combined gas chromatographic-mass spectrometric analysis of the inhibitors showed them to be flavonoids. (+)-Catechin was identified in the fraction eluted 5 min after injection that also showed maximum inhibition. Other flavonoids such as epicatechin, quercetin and naringenin also inhibited calmodulin-promoted phosphodiesterase activity. Among the phenolic compounds commonly encountered in plant tissue only caffeic acid inhibited calmodulin-promoted phosphodiesterase activity. Inhibition by catechin and caffeic acid could be reversed by increasing the calmodulin concentration in the assay mixture. Both catechin and caffeic acid inhibited Ca- and calmodulin-promoted phosphorylation of soluble proteins from corn coleoptiles. The physiological properties of flavonoids are discussed in light of this evidence.     

The presence of citrulline in epidermal proteins

Citrulline is present in the stratum corneum proteins of human, cow snout, pig snout and guinea pig epidermis but is absent from the stratum corneum proteins of frog, mouse, turtle, rat and hamster epidermis. The amino acid is released by acid hydrolysis and ranges from 1.7 to 5.5 residues per thousand residues of protein amino acid. Protein derived citrulline co-chromatographs with authentic L-citrulline on an amino acid analyzer, on Dowex-50, on Dowex-2 and on thin-layer chromatography. Dansylated material co-chromatographed with authentic dansyl-L-citrulline in two thin-layer chromatography systems. Labelling experiments have shown that the protein bound citrulline is derived from protein bound arginine and probably results from enzymatic conversion of the guanido group to the ureido group.     

Preparative isolation of polyphosphoinositide fractions from ox brain

A simple preparative method for chromatographic isolation of pure fractions of di- and triphosphoinositides (1-phosphatidylinositol 4-phosphate and 1-phosphatidylinositol 4,5-bisphosphate) from ox brain is described. Polyphosphoinositide fractions have been obtained by ion-exchange chromatography of the lipid extract using gradient elution with 0-0.6 M ammonium acetate in chloroform/methanol/water (20:9:1) from a DEAE-cellulose column. Before chromatography, divalent metal ions were removed from the lipid extract by passing through a Dowex-50 (H+) column and lipids were converted to the sodium salt by neutralisation with sodium hydroxide in methanol solution. After chromatography, fractions of di- and triphosphoinositides were precipitated in methanol/water mixture (1:1) by evaporation in a vacuum to a final concentration of about 4 M ammonium acetate. Necessary salts of di- and triphosphoinositides were obtained by passing the ammonium salts of the lipids through Dowex-50 (H+) and neutralising with corresponding base in methanol solution. About 0.35 mmol of diphosphoinositide and 0.63 mmol of triphosphoinositide were obtained from 1 kg of wet ox brain tissue.     

Changes in acid soluble nucleotide components of candida utilis during the cell cycle

Acid soluble extracts obtained at 30 min intervals from cells of C. utilis growing in synchrony in a phased culture (cycle time 51/2 hr) were fractionated on a Dowex-1-formate column. The series of fractionation profiles showed changes in number and amounts of components over the cell cycle. Transient accumulations of numerous components over the complex pool were observed. The significance of the changes are discussed in relation to practical applications and cell metabolism.     

The value of Dowex-50 in fractionation of nucleotides from acid soluble pool. A caution

Problems were encountered in separating acid soluble nucleotides into four main groups by chromatography on Dowex-50(H⁺) columns. These difficulties derived from the low affinity of ATP, ADP, GDP, GTP, CDP, and CTP for Dowex-50 (H⁺) ion exchanger under mild acid conditions. Thus, under the conditions described, these compounds travel together with uridine compounds.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

During the course of the investigation on the production of nucleotide by fermentative processes, it was found that a large amount of ATP and ADP or GTP and GDP, in addition to a smaller amount of AMP or GMP, accumulated in the culture broth when Brevibacterium ammoniagenes ATCC 6872 was incubated in a medium containing adenine or guanine.

After treatment of the culture filtrate with charcoal, the nucleotides were isolated by ion-exchange chromatography on Dowex-1 × 2 (Cl^?-form). They were identified by paper-chromatography, ultraviolet absorption spectra and analyses of base, ribose and phosphate. The ATP preparation from the broth had the same activity with that of authentic sample in the β-aspartokinase system from Corynebacterium glutamicum.     

5-Bromouridylyl-(3′ → 5′)-Adenosine Isolated from 5-Bromouracil-Induced Filaments of Escherichia coli

A dinucleoside monophosphate was isolated from 5-bromouracil-induced filaments of a thymine auxotroph of Escherichia coli K-12. The dinucleoside monophosphate was fractioned from a [(14)C]5-bromouracil-labeled perchloric acid extract using Dowex-1-formate ion-exchange chromatography. Sephadex chromatography revealed its molecular weight to be 710. Snake venom phosphodiesterase digest of the dinucleoside monophosphate yielded [(14)C]5-bromouridine and adenosine 5'-monophosphate. The presence of [(14)C]5-bromouracil in bacterial ribonucleic acid indicates that ribonucleic acid, which had incorporated 5-bromouracil, was the probable source of this dinucleoside monophosphate, 5-bromouridylyl-(3' --> 5')-adenosine.     

Specificity of gamma-glutamyl cyclotransferase.

Using the phthaloyl method, 18 gamma-L-glutamyl peptides labelled with 14-C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified gamma-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that gamma-glutamyl cyclotransferase acts only on the L-form of gamma-glutamyl substrates; the enzyme could, therefore, be used for preparation of gamma-D-glutamyl peptides from their racemic mixtures. The specificity of gamma-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as gamma-L-glutamyl-NH--CHR--COOH. The amino acid linked to the gamma-glutamyl group must be in the L configuration.     

Production of Coenzyme A by Sarcina lutea

To develop an efficient method for the production of coenzyme A (CoA), optimal conditions for its formation from pantothenic acid, cysteine, and adenine were studied. A number of microorganisms were screened for production of CoA. Strains belonging to the genera Sarcina, Bacillus, Microbacterium, Micrococcus, and Serratia accumulated CoA. Among these, Sarcina lutea was selected as the best organism, and the culture conditions for the production of CoA were investigated with this organism. Under optimal conditions, 600 mug of CoA per ml was accumulated in the culture broth. CoA was readily isolated in high purity by the use of charcoal, diethylaminoethyl-cellulose, Sephadex G-25, and Dowex-50. Yields of isolated CoA were over 33% from culture broth.     

Determination of argininosuccinate in normal blood serum and liver

Argininosuccinate has been determined in normal serum and liver extract of rats by chromatographic analysis on Dowex-1-acetate after a brief period of in vivo labeling with l-[U-¹⁴C] citrulline. The amounts found were within the range of other amino acid intermediates of the ornithine cycle, determined in the same samples. Both ¹⁴C and ninhydrin color were measured. Chromatography on Amberlite columns, subsequent to fractionation on Dowex, allowed determination of the other amino acids. Fractionation on Dowex-1-acetate provides a reliable and highly selective method for the analysis of minute amounts of argininosuccinate in biological samples containing other amino acids.     

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.     

Production of a precursor to the pyrimidine moiety of thiamine.

The supernatant fluid from cultures of Escherichia coli W-11, a pur E mutant, prevented the inhibition of growth of E. coli B in a medium containing adenine or adenosine. Adenine inhibition was prevented more readily than adenosine inhibition. More than 90% of the biological activity of the supernatant fluid was recovered in the anionic fraction after treatment with Dowex-50 (NH4+). The cationic fraction, containing large amounts of 5-aminoimidazole ribonucleoside (AIRS), did not prevent adenine inhibition. The W-11 supernatant fluid was shown by bioautography to contain only one compound that prevented adenine inhibition. Proliferating and non-proliferating cultures produced only one compound that prevented adenine inhibition. The compound was shown to be an intermediate (int-1) in the biosynthesis of the pyrimidine moiety of thiamine, Int-1 was stable during sterilization at 121 C for 15 min, during concentration by either flask evaporation or lyophilization, and after storage for several days at 4 C or at -- 20 C. Int-1 was distinguishable from other known derivatives or intermediates of the pyrimidine moiety. A scheme is presented that illustrates the proposed relationship between int-1 and the synthesis of thiamine.     

Purification of commercial preparations of NADP+ from AMP contamination

A method for purification of commercial preparations of NADP+ from AMP contamination is described. The purification procedure includes one-step anion-exchange chromatography on Dowex-1 (formate) and results in a highly purified salt-free coenzyme with a yield of 70-80%. The chromatography conditions have been selected allowing for complete separation of AMP from NADP+ in a HCOOH concentration gradient. This is followed by NADP+ elution with 1.5 M HCOOH containing HCOOK at a concentration at which the salt remains in solution during subsequent precipitation and washing of NADP+ with acetone. An addition of HCOOK is necessary to reduce the coenzyme elution volume that is important for further precipitation of NADP+ with acetone.     

Cyclic adenosine monophosphate (cAMP) production in avian theca cells during follicular maturation

LH was used to stimulate cAMP production in theca cells from the 5 largest preovulatory follicles of hens and this was related to LH-stimulated androstenedione production in the same cells. cAMP production was stimulated by LH to the same extent in theca cells from each follicle. However, LH was not effective in stimulating androstenedione production in theca cells from the largest follicle (T1), although androstenedione production was greatly increased by LH in the smaller follicles (T2-T5). Effects similar to those of LH on cAMP production were observed in response to forskolin, indicating that the intrinsic adenylate cyclase activity was similar in theca cells from each follicle. In addition, forskolin was unable to stimulate androstenedione production by T1 cells. Our results provide evidence that the levels of receptor-mediated and non-receptor-mediated cAMP production are similar in theca cells from the 5 largest follicles. We conclude that the step that restricts the ability of T1 cells to produce androgen is distal to cAMP generation.     

ACID-SOLUBLE NUCLEOTIDES OF CHLAMYDOMONAS MOEWUSII, WILD TYPE AND A PARALYZED MUTANT

1. An attempt was made to correlate flagellar paralysis in theChlamydomonas mutant M. 1002 with some change in nucleotidemetabolism.
2. Cells of this mutant did not differ significantlyfrom wildtype in their ATP content.
3. Perchloric acid extractsof wild-type and mutant cells werecompared by gradient formateelution chromatography from Dowex-1columns.
4. Less nucleotidewas extracted from mutant cells than from wildtypecells.
5. The30-minute extracts of mutant cells contained a fraction(peak"B") absent from similar extracts of wild-type cells.This fractioncontained hypoxanthine, guanine, and an unidentifiedUV-absorbingcomponent.
6. It thus appears that flagellar paralysis in thiscase may becorrelated with an impairment in the metabolismof purine compounds.

(Received June 24, 1965; )     

Ubiquitin-Specific Peptidase 5, a Target Molecule of Vialinin A,Is a Key Molecule of TNF-α Production in RBL-2H3 Cells

Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.