Detection of cystathionine and lanthionine ketimines in human urine

A recently developed HPLC procedure for the determination of cystathionine ketimine (CK) and lanthionine ketimine (LK) has been applied to the detection of these compounds in human urine. The assay has taken advantage of the selective production of an absorbance at 380 nm, not seen with other amino acids, when the two ketimines are reacted with phenylisothiocyanate. Coelution with authentic phenylthiohydantoin derivatives of CK and LK and the identical absorption spectra establish the identity of the compounds found in the urine with the synthetic products. Quantitation of the two ketimines by HPLC indicates that the excretion of CK and LK is respectively 606 micrograms and 84 micrograms per g of creatinine as mean values of 10 healthy subjects of both sexes, 20-40 years old, in the early morning voided urine.     

Inhibition of early steps of de novo fatty-acid biosynthesis by different xenobiotica

The importance of the early steps of de novo fatty-acid biosynthesis is discussed in terms of rate-limiting enzymic reactions with respect to their inhibition by xenobiotics. The inhibitory spectra of allicin as an inhibitor of the acetyl-CoA-synthase, two classes of graminicides (cyclohexane-1,3-diones and aryloxyphenoxypropionic acids) as inhibitors of acetyl-CoA-carboxylase, and the two antibiotics cerulenin and thiolactomycin, which affect the condensing step in fatty-acid biosynthesis, are compared.     

Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).     

Similarity of the oxidation products of L-cystathionine by L-amino acid oxidase to those excreted by cystathioninuric patients

L-Cystathionine is oxidized by snake venom L-amino acid oxidase at a rate about half that with L-leucine at pH 8.5. The appearance of an absorbance at 296 nm and quantitation of the products of oxidation in the presence of catalase indicate formation in the solutions of a seven-membered ketimine ring produced by cyclization of the monoamino monoketo derivative of cystathionine. A limited double deamination has also been observed. In the absence of catalase, S-(carboxymethyl)homocysteine and S-(beta-carboxyethyl)cysteine have been identified together with ninhydrin-unreactive compounds yielding the above mentioned carboxy compounds upon hydrolysis with HCl. Authentic samples of the monoamino monoketo analogs of cystathionine have been prepared and compared with the enzymatic products. Cyclization of the synthetic products into the ketimine ring is pH-dependent as established by UV spectrum and other assays. Compounds derived from either the oxidation or the reduction of the ketimine have been prepared. It was found that many products of enzymatic and chemical changes of cystathionine and its ketimine described in the present paper are identical with those identified in the urine of cystathioninuric patients. This result indicates the occurrence in humans of secondary metabolic routes of cystathionine centered on the production of cystathionine ketimine, in equilibrium with the open form, which in cystathioninurics is revealed by the lack of cystathionase.     

Armaments and ornaments: an evolutionary explanation of traits of dual Utility

Secondary sexual characters in many species function both in male-male competition and as cues for female choice. Based on a literature compilation of existing knowledge of traits with this dual function, we propose that they commonly arise through intersexual selection processes and serve as honest signals to other males regarding fighting ability or dominance. Faking these traits, here called armaments, (i.e. weapons and status badges) is difficult, as they are constantly put to trial in male-male contests. Females that subsequently utilize them as indicators of male phenotypic quality when selecting a partner will benefit by acquiring males of higher quality to father their offspring. Thus, evolution of armaments through male-male competition is seen as a usually initiating process, whereas female choice later may assume a role as an additional selective factor. The reverse, that males use information from traits evolved through female choice, is, however, also possible. The traditional view of independently evolved and temporarily unordered intra- and intersexual selection processes fails to explain dual trait functions. Moreover, our model may more satisfyingly than traditional ones explain how trait honesty and trait genetic variance are maintained: theoretical and empirical evidence suggests that such honesty and variation are more easily maintained under male-male competition than under female choice.     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.     

N-dealkylation of chlorimipramine and chlorpromazine in patients undergoing chronic treatment with both drugs: preliminary results

Data obtained in this preliminary study show that in patients chronically treated either with Chlorimipramine (n = 6) or with Chlorpromazine (n = 2), significant amounts of the corresponding Nor1- and Nor2-metabolites were detected in plasma. The role of these metabolites in the overall therapeutic effect is under investigation.     

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (greater than 96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.