Increase in cystathionine content in rat liver mitochondria after D,L-propargylglycine administration

Summary Intraperitoneal administration of D,L-propargylglycine to rats resulted in an increase in the cystathionine content of whole liver and liver mitochondria. Cystathionine in mitochondria was identified by amino acid analysis, thin layer chromatography, high-voltage paper electrophoresis and liquid chromatography-mass spectrometry. The cystathionine content of whole liver was 5.37 ± 1.59µmol per g of fresh liver at 14 h after the administration of 50 mg of D,L-propargylglycine per kg of body weight, while 0.07 ± 0.02µmol of cystathionine per g of fresh liver was detected in the control rats. The cystathionine content of liver mitochondria from both groups of rats was 9.40 ± 1.20 and 0.19 ± 0.04 nmol of cystathionine per mg of protein, respectively. The mitochondrial cystathionine increased dose-dependently with the increase of D,L-propargylglycine administered. The increase was proportional to the time after the administration up to 12 h, and then decreased. The increase of cystathionine in the liver mitochondria was linearly proportional to that in the whole liver. These results suggest that cystathionine in liver mitochondria is in an equilibrium with that in the cytosol.     

Identification ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid with a metabolic intermediate betweenS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine andS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid

Summary S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid (I) was chemically synthesized in 15% yield by incubating a reaction mixture oftrans-urocanic acid and 3-fold excess of 3-mercaptopyruvic acid at 45°C for 6 days. The synthesized compound was characterized by fast-atom-bombardment mass spectrometry and high-voltage paper electrophoresis. CompoundI was identified with a product of an enzymatic reaction ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine (II) with rat liver homogenate in a phosphate buffer, pH 7.4. CompoundI was degraded toS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid (III), a compound previously found in human urine [Kinuta et al. (1994) Biochem J 297: 475–478], by incubation with rat liver homogenate. From these results, we suggest that compoundI is a metabolic intermediate for the formation of compoundIII from compoundII. The present pathway follows a formation of compoundII fromS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl] gluthathione [Kinuta et al. (1993) Biochim Biophys Acta 1157: 192–198], a proposed metabolite ofl-histidine.     

Facile synthesis of (R)N-2-hydroxyacyl-L-cysteine derivatives: (R)N-2-hydroxyacyl transfer from enzymatically-synthesized (R)S-2-hydroxyacylglutathione derivatives to L-cysteine

Summary N-(R)-2-Hydroxyacyl-L-cysteine derivatives were conveniently synthesized by the reaction of the corresponding S-(R)-2-hydroxyacyl-glutathione with cysteine. The (R)2-hydroxyacyl group was transferred from the S-glutathionyl moiety to S-cysteinyl, forming the corresponding (R)S-2-hydroxyacylcysteine; this rearranged to the (R)N-hydroxyacylcysteine. These compounds have anti-proliferative activity associated with the inhibition ofde novo pyrimidine synthesis.Abbreviations TRIS tris(hydroxymethyl) aminomethane - DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

Changes in amino acid decarboxylation in maize (Zea mays; Poaceae) tissues in response to bird cherry-oat aphid (Rhopalosiphum padi; Aphididae) infestation

The study was focused on changes in the activities of ornithine decarboxylase (ODC), lysine decarboxylase (LDC) and tyrosine decarboxylase (TyDC) in maize seedlings infested by bird cherry-oat aphid (Rhopalosiphum padi L.).Obtained results showed that the aphid infestation induced LDC activity strongly within tissues of less aphid-settled maize (Waza cv.) during the first week of the infestation in comparison to the control plants and the activity was suppressed after two weeks. However, TyDC activity fluctuated under the aphid infestation. In relation to the control, the enzyme activity was inhibited on the first day, activated after the first week and reduced again two weeks into the experiment. A significant reduction in ODC activity was also observed in seedlings of aphid-infested maize Waza cv. during the first week. In a more susceptible maize cv. (Złota Karłowa), an increase in LDC and TyDC activities in relation to the control and a simultaneous decrease in ODC activity were noted after the first day of the infestation. After one week of aphid attack, TyDC activity was induced and ODC was inhibited, whereas after two weeks ODC activity was decreased with a simultaneous increase in LDC activity in the Złota Karłowa seedlings.     

In vitro Dimroth rearrangement of 1-(2-carboxyethyl) adenine to N6-(2-carboxyethyl)adenine in single-stranded calf thymus DNA.

The new adduct N6-(2-carboxyethyl)adenine (N6-CEA) was prepared from 1-(2-carboxyethyl)adenine (1-CEA) by base catalyzed (Dimroth) rearrangement of 1-CEA. The structure of N6-CEA was assigned on the basis of UV spectra and electron impact and isobutane chemical ionization mass spectra. When the carcinogen beta-propiolactone was reacted in vitro with calf thymus DNA, 1-CEA but not N6-CEA was detected on paper chromatograms following acid hydrolysis of the DNA. When BPL-reacted single-stranded DNA was incubated at pH 11.7 (37 degrees C, 18 h) prior to acid hydrolysis, it was found that 1-CEA was completely converted to N6-CEA in DNA by Dimroth rearrangement, whereas no conversion occurred at pH 7.5. The extent of Dimroth rearrangement at various pHs and temperatures was determined for 1-CEA, 1-methyladenine (1-MeA), 1-(2-carboxyethyl)-deoxyadenosine-5'-monophosphoric acid (1-CEdAdo5'P) and the phosphodiester 5'-O-(2-carboxyethyl)phosphono-1-(2-carboxyethyl)deoxyadenosine (1-CE-Ado-5'-P-CE).     

Synthesis of bis-(methyl 3,4,6-tri-O-acetyl-D-glucopyranosid-2-yl)-oxamides

The synthesis of a new bis-(D-glucopyranosid-2-yl)oxamides via the key intermediate, N-acetyl N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl) oxamic acid chloride (2alpha) is described. Treatment of compound 2alpha with methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranoside afforded N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl)-N'-(methyl 3,4,6-tri-O-acetyl-beta-D-glucopyranosid-2-yl)-oxamide. Reaction of 2alpha with 1,2-diaminoethane afforded 1,2-bis-[N,N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)]ethyloxamide as a main product, while 2-N-[N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)oxamide]-ethyl acetamide was formed as a side product. Reaction of 2alpha with 1,3-diamino-2-hydroxypropane gave only 1,3-bis-N,N-[N'-(methyl 3',4',6'-tri-O-acetyl-2'-deoxy-alpha-D-glucopyranosid-2'-yl)-oxamido]-2-propanol.     

Conscientious metabolic monitoring on a patient with hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome undergoing anaesthesia

Summary. Currently we know not more than 50 patients who show an interesting combination of increased plasma ornithine concentrations, postprandial hyperammonemia, and homocitrullinuria (HHH-syndrome). Since exact knowledge of this severe, although rare syndrome is important for any perioperative or intensive medical treatment concerning therapy and progression of the disease, we report a comprehensive study on a 32-year old woman with this rare multifaceted disorder who had to undergo general anaesthesia. For the first time amino acid status in plasma, urine, cerebrospinal fluid and especially polymorphonuclear leucocytes, which in the investigation showed to be valuable tool for evaluating amino acid metabolism in nucleated cells in HHH-syndrome, and further important pathophysiologic indicators of cellular and metabolic function have been conscientiously investigated and compared. The pathophysiological repercussions of our results as well as the recommendations for conscientious therapeutical management are discussed. Received September 9, 1999 Accepted July 1, 2000     

A porcine animal model to mimic the restart of enteral nutrition (refeeding-model)

With the aim towards establishing an animal model of total parenteral nutrition (TPN), 12 piglets aged 9 weeks (mean body weight 21 kg) were surgically provided with central venous catheters. Six piglets were nourished parenterally with the objective to reach a 14-d period of TPN; the other six piglets served as control and were fed normally. Only one animal from each group could be monitored over the whole period. Nine piglets were euthanised on d 13 and one on d 12. No animal showed fever or signs of septicaemia during the study. The levels of Ca, Mg, Na and P in the blood were within the normal range as were those for blood glucose and plasma creatinine. Symptoms of the TPN included: transient diarrhoea, occasional appearance of faecal blood and occasional absence of defecation. A reduced small intestine length and altered mucosal morphology and function were observed. One animal showed bile stasis at the end of the study. All TPN animals showed a remarkably high level of blood urea early in the morning. The intestinal symptoms observed may resemble the human situation during TPN. However, due to the fast growth rate, pigs aged 9 weeks have higher nutrient requirements per kg body weight. Consequently, the osmolality of the nutrient solution was necessarily high. Whether the significantly higher blood urea observed in the TPN group reflected a catabolic metabolism during the starving period at night-time could not be conclusively shown. Alternatively, it could reflect a slower growth rate and a resulting quantitative excess of amino acids (AA), or could have been the consequence of a suboptimal AA composition. A permanent infusion would be favourable in order not to overcharge the capacity for glucose uptake and amino acid metabolism during the infusion.     

Differential diagnosis of (inherited) amino acid metabolism or transport disorders

Summary Disorders of amino acid metabolism or transport are most clearly expressed in urine. Nevertheless the interpretation of abnormalities in urinary amino acid excretion remains difficult. An increase or decrease of almost every amino acid in urine can be due to various etiology. To differentiate between primary and secondary aminoacido-pathies systematic laboratory investigation is necessary. Early diagnosis of disorders of amino acid metabolism or transport is very important, because most of them can be treated, leading to the prevention of (further) clinical abnormalities. In those disorders, which cannot be treated, early diagnosis in an index-patient may prevent the birth of other siblings by means of genetic counseling and prenatal diagnosis.Primary aminoacidopathies can be due to genetically determined transport disorders and enzyme deficiencies in amino acid metabolism or degradation. Secondary aminoacidopathies are the result of abnormal or deficient nutrition, intestinal dysfunction, organ pathology or other metabolic diseases like organic acidurias.A survey of amino acid metabolism and transport abnormalities will be given, illustrated with metabolic pathways and characteristic abnormal amino acid chromatograms.     

Identification of an oxidation product of aminoethylcysteine ketimine dimer

Summary. In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine dimer by oxygen reactive species, we give here data for the identification of the α, β unsaturated sulfoxide as the main product of interaction of the dimer with H₂O₂. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography. Received March 24, 1998, Accepted October 20, 1998     

Oxidative deamination of Se-(1-carboxyethyl)-,Se-(1-carboxypropyl)- and Se-(2-carboxyethyl)-selenocysteine by snake venom L-aminoacid oxidase

Details are reported for the synthesis of Se-(1-carboxyethyl)-selenocysteine (1-CESeC), Se-(1-carboxypropyl)-selenocysteine (1-CPSeC) and Se-(2-carboxyethyl)-selenocysteine (2-CESeC). They can be obtained in pure cristalline form with good yield. Some chromatographic properties, useful for their identification, are described. The three aminoacids are good substrates for snake venom L-aminoacid oxidase, giving the corresponding alpha-ketoacids as reaction products.     

Synthesis and structures of copper and gold complexes of the P,N ligands RNC(Bu)C(H)RPPh2 (R = SiMe3, H)

The reaction of the chelating P,N ligand RNC(Bu^t)CH(R)PPh₂ (R = SiMe₃) (1) with CuCl and CuCl₂ (probably by way of reduction to Cu(I) by the phosphine ligand) or Cu(NCCH₃)₄ClO₄ yielded the dimeric 1:1 complex [Cu{PPh₂CH(R)C(Bu^t)NR}Cl]₂ (2) or the monomeric 2:1 complex [Cu{PPh₂CH(R)C(Bu^t)NR}₂]ClO₄ (3), respectively. The presence of trace amounts of water during the reaction resulted in the successive cleavage of the two trimethylsilyl groups of the ligand and the formation of the monomeric chelate complexes [Cu{PPh₂CH(R)C(Bu^t)NH}₂]ClO₄ (4) and [Cu{PPh₂CH₂C(Bu^t)NH}₂]ClO₄ (5). Oxidation of 5 by atmospheric oxygen led to small quantities of the blue Cu(II) complex [Cu{(O)PPh₂CH₂C(Bu^t)NH}₂](ClO₄)₂ (6). The dimeric gold complexes [Au{PPh₂CH₂C(Bu^t)NH}]₂X₂ (X = BF₄, ClO₄) (7) were similarly obtained from the previously described Au{PPh₂CH(R)C(Bu^t)NR}Cl by replacing the covalently bound chlorine with the weakly coordinating anions in the presence of small quantities of water. The solution and solid state structures (except 5) of all complexes were determined by NMR spectroscopy and X-ray crystallography.     

Synthesis of non-proteinogenic (D)- or (L)-amino acids by asymmetric hydrogenation

Summary Non-proteinogenic amino acids play an increasing role in oligopeptide chemistry. Their pharmacological and chemical properties, caused by D-configuration and unnatural residues, are more and more used for drug design. Different methods of asymmetric synthesis have been developed during the last decade to prepare unusual amino acids. One of them, the asymmetric hydrogenation of dehydroamino aids catalyzed by chiral rhodium (I) complexes, will be described. A series of examples, D- and L-configured, like naphthyl-, thienyl-, furyl-, and pyridylalanines, as well as phenylalanines substituted by chlorine, fluorine, p-nitro, p-methyl, p-trifluoromethyl, p-isopropyl, and p-tert-butyl have been prepared and characterized. Some analytical data like melting points and values of optical rotation are summarized in tables.Abbreviations (–)-DIOP (4R,5R)-4,5-Bis(diphenylphosphinomethyl)-2,2-dimethyl-1,3-dioxolane - (–)-BPPM (2S,4S)-N-tert-Butoxycarbonyl-4-diphenylphosphino-2-diphenylphosphinomethyl-pyrrolidine - Ph--glup Phenyl 4,6-O-(R)-benzylidene-2,3-O-bis(diphenylphosphino)--D-glucopyranoside - DuPHOS 1,2-bis-(phospholano)benzene - PROPRAPHOS 2,3-O,N-bis(diphenylphosphino)-1-(naphthoxy)-2-hydroxy-3-isopropylamino propane - PINDOPHOS 2,3-O,N-bis(diphenylphosphino)-1-(4-indolyloxy)-2-hydroxy-3-isopropylamino propane     

5-(pyrrolidin-2-yl)-1H-tetrazole and 5-[(pyrrolidin-2-yl)methyl]-1H-tetrazole: proline surrogates with increased potential in asymmetric catalysis

Catalytic enantioselective methodology has dramatically been enriched by the re-discovery of the simple amino acid proline as a chiral catalyst in the year 2000. Although no catalyst offers such a simple and broad access to quite complex reaction products, as does proline, its synthetic potential is not unrestricted, what is especially connected to its poor solubility in organic media. Exchange of the carboxylic moiety by a tetrazole unit leads to proline surrogates, that by far outperform proline with respect to yield, enantioselectivity, reaction time, substrate and solvent scope, catalyst loading, and stoichiometry of the compounds used in excess. These factors are discussed and critically compared with selected representative proline-catalyzed reactions.     

Kainic acid (KA)-induced seizures in Sprague-Dawley rats and the effect of dietary taurine (TAU) supplementation or deficiency

Summary Male Sprague-Dawley rats received TAU supplementation (1.5% in drinking water) or TAU deficient diets for 4 weeks to test for a possible neuroprotective role of TAU in KA-induced (10 mg/kg s.c.) seizures. TAU supplementation significantly increased serum and hippocampal TAU levels, but not TAU content in temporal cortex or striatum. TAU deficient diets did not attenuate serum or tissue TAU levels. Dietary TAU supplementation failed to decrease the number or latency of partial or clonic-tonic seizures or wet dog shakes, whereas a TAU deficient diet decreased the number of clonictonic and partial seizures. This study does not support previous observations of an anticonvulsant effect of TAU against KA-induced seizures. KAtreatment decreased ₂-adrenergic receptor binding sites and TAU content in the temporal cortex across all dietary treatment groups, supporting previous evidence of severe KA-induced damage and neuronal loss in this brain region.     

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine in normal human urine

Summary A compound, which had the same mobility on a high-voltage paper electrophoretogram and the sameR _F value on a thin-layer chromatogram as those ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (I), was partially purified from human urine by ion-exchange column chromatography. The compound gave a signal at m/z 260 on its FAB mass spectrum, which was assigned as MH⁺ of compound I. These results suggest that the urinary compound is compound I and it is a physiological precursor of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid [Kinuta et al., (1991) Biochem J 275: 617–621].     

Synthesis of new 2-C-(2,3:5,6-di-O-isopropylidene)-beta-D-mannofuranosyldithioacetate derivatives

Various new C-glycosides have been synthesized through the thioacylation reaction of different amines by a 2-C-mannofuranosyldithioacetate. Amino acids, di- and polyamines (putresceine, spermine, spermidine) and one amino alcohol were in particular used to generate glycothiopeptidic or precursors of "bola" structures.     

Phosphodiesterolytic activity of lanthanide (III) complexes with α-amino acids

Kinetics of the hydrolysis of BNPP (bis(4-nitrophenyl)phosphate) mediated by lanthanide - samarium (III) and ytterbium (III) - alone and in the presence of various alfa amino acids has been systematically studied at 37.0 °C and I = 0.15 M in NaClO₄, in the pH interval of 7-9. The rate of BNPP cleavage is sensitive to metal ion concentration, pH, and ligand to metal molar ratio. Hydrolysis follows Michaelis-Menten-type saturation kinetics. For both metals, high pH values markedly increase the observed activity. Besides, potentiometric titrations of all these systems under identical conditions allowed us to identify the active coordination compounds towards hydrolysis. The results show that complexes with phosphodiesterolytic activity are monomeric cationic species such as [Ln(aa)₃(OH)]²⁺ or [Ln(aa)₂(OH)₂]⁺. Since phosphodiesterolytic activity is evident above pH 7 and it is increased with increasing pH, hydrolytic reactions of the metals are competitive processes that could lead to their precipitation as Ln(OH)₃(s). In this sense, ligand excess (for example, ligand to metal molar ratio equal to 30) was employed. Furthermore, due to its more extended hydrolysis, ytterbium shows, in general, less activity than samarium under the studied conditions. In general, a good phosphodiesterolytic activity is observed for these complexes under similar conditions to the physiological ones. Amino acids could be easily derivatized without changing their coordinating ability, leading to lanthanide complexes possibly capable of efficiently hydrolyzing the phosphodiester linkages of nucleic acids.     

Structural and mechanistic studies on N-(2-carboxyethyl)arginine synthase

N²-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the β-lactamase inhibitor clavulanic acid in Streptomyces clavuligerus. Crystal structures of tetrameric CEAS-ThDP in complex with the substrate analogues 5-guanidinovaleric acid (GVA) and tartrate, and a structure reflecting a possible enol(ate)-ThDP reaction intermediate are described. The structures suggest overlapping binding sites for the substrates d-glyceraldehyde-3-phosphate (d-G3P) and l-arginine, and are consistent with the proposed CEAS mechanism in which d-G3P binds at the active site and reacts to form an α,β-unsaturated intermediate, which subsequently undergoes (1,4)-Michael addition with the α-amino group of l-arginine. Additional solution studies are presented which probe the amino acid substrate tolerance of CEAS, providing further insight into the l-arginine binding site. These findings may facilitate the engineering of CEAS towards the synthesis of alternative β-amino acid products.     

The distribution of nitrate reductase in tomato (Lycopersicon esculentum) leaves as affected by age

The distribution of nitrate reductase (NR, EC 1.6.6.1.) in the leaves of single-stem tomato plants ( Lycopersicon esculentum Mill., cv. Vandenbergs Moneydor) was studied using an in vitro test. The activity decreased from young to old leaves. However, a low value (NR minimum) occurred in some leaves below the apex, usually in the almost completely expanded leaves, provided that the plants received sufficient nitrate to induce optimum NR activity in all the leaves. When insufficient nitrate was available there was NR in the young leaves only. The observed NR minimum coincided with a low value for soluble carbohydrates and amino acids. Since there was no extra export of labelled carbon from the leaves with the NR minimum, it is suggested that in the almost completely expanded leaves carbohydrates produced by photosynthesis are mainly used for the production of polysaccharides for new cell walls. Consequently, less are left for the production of keto acids, which can act as acceptors for reduced nitrogen. Therefore, less amino acids are produced, and this may result in a lowered protein synthesis, including a lowered synthesis of nitrate reductase.