The serum level of certain aminoacids in chronic liver diseases associated with impaired oral glucose tolerance.

We studied the plasma levels of some aminoacids and immunoreactive insulin in chronic liver diseases with impaired glucose tolerance during oral glucose tolerance test (OGTT). There are significant alterations of aminoacids levels and insulin in chronic hepatitis and liver cirrhosis, especially followed by impaired glucose tolerance.

G20210A prothrombin gene mutation identified in patients with venous leg ulcers.

The G20210A mutation variant of prothrombin gene is the second most frequent mutation identified in patients with deep venous thrombosis, after factor V Leiden. The risk for developing deep venous thrombosis is high in patients identified as heterozygous for G20210A mutation. In order to identify this polymorphism in the gene coding prothrombin, the 345bp fragment in the 3'- untranslated region of the prothrombin gene was amplified using amplification by polymerase chain reaction and enzymatic digestion by HindIII (restriction endonuclease enzyme). The products of amplification and enzymatic's digestion were analized using agarose gel electrophoresis. We investigated 20 patients with venous leg ulcers and we found 2 heterozygous (10%) for G20210A mutation. None of the patients in the control group had G20210A mutation. Our study confirms the presence of G20210A mutation in the Romanian population. Our study also shows the link between venous leg ulcers and this polymorphism in the prothrombin gene.

Mutation spectrum and phenylalanine hydroxylase RFLP/VNTR background in 44 Romanian phenylketonuric alleles.

The mutation spectrum and polymorphic haplotype background in 22 Romanian families have been analysed in this study using the restriction digestion of phenylalanine hydroxylase (PAH) regions specifically amplified or the DGGE/direct sequencing methods. Eleven PAH mutations specifically associated with six mutant haplotypes were detected. In spite of the relative heterogeneity of the molecular defects in the PAH gene, three mutations covered almost 70% of all alleles: R408W, 47.72%, 21/44; K363fsdelG 13.63%, 6/44; and P225T 6.81%, 3/44. Among these, R408W, the most frequent mutation in our population, represented 50% of all the phenylketonuric (PKU) chromosomes. Splice mutation IVS12nt1g-->a affected two PAH alleles (4.54%); the remaining seven mutations were rare, each having an effect on just one chromosome (1/44), resulting in a relative frequency of 2.27%. A high frequency was observed in our PKU samples for the relatively uncommon mutations, K363fsdelG and P225T mutation, suggesting a possible founder effect at origin. Within the investigated panel, these mutations, both very rare among other Caucasians were exclusively linked to haplotype 5.8 and 1.7, respectively. These results provide a basis for the development of a routine molecular analysis of Romanian PKU families.

Enzymatic properties of 8-bromoadenine nucleotides.

8-Bromoadenine nucleotides were tested as potential substrates and/or inhibitors of mitochondrial processes in intact or disrupted organelles, as substrates of various phosphotransferases, and as allosteric effectors in the reactions catalyzed by phosphofructokinase, isocitrate dehydrogenase, glutamate dehydrogenase, and fructose-1,6-bisphosphatase. 8-BrATP and 8-BrADP are not recognized by the translocase system located in the inner mitochondrial membrane and cannot be used as usbstrates in oxidative phosphorylation and related reactions catalyzed be beef heart submitochondrial membranes. This confirms the high specificity for adenine nucleotides of the mammalian systems involved in energy-yielding and energy-requiring reactions. However, 8-BrATP and 8-BrADP are able to substitute for the natural adenine nucleotides in reactions catalyzed by many phosphotransferases, although their capacity as phosphate donors and acceptors is generally much reduced. On the other hand, in almost all investigated cases, the 8-bromoadenine nucleotides have lost the capability of the natural adenine nucleotides to act as allosteric effectors, indicating that the structural requirements for allosteric activity are more stringent than those for catalytic activity.

Nucleotide specificity of pyruvate kinase and phosphoenolpyruvate carboxykinase.

Various analogues of adenosine 5'-diphosphate with modifications in the heterocyclic base residue were tested as substrates of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC. 2.7.1.40) and guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). The significance of different structural elements for the enzyme-substrate interaction is discussed. While pyruvate kinase shows a rather broad specificity for these analogues, phosphoenolpyruvate carboxykinase has a more stringent requirement for nucleotides, the intact keto and NH groups at C6 and N1 of the pyrimidine ring representing essential sites for the phosphoenolpyruvate carboxykinase substrate interaction. The biological significance of the different substrate specificities of pyruvate kinase and phosphoenolpyruvate carboxykinase is discussed as a possible metabolic control factor.

Structural and enzymatic properties of adenine 1-oxide nucleotides.

We decribed the preparation of adenine 1-oxide nucleotides by oxidation of the natural compounds with monopermaleic acid in aqueous solutions at neutral pH, with an overall yield after chromatographic purification between 75 and 80%. If irradiated, the adenine 1-oxide nucleotides undergo a photochemical rearrangement reaction, the main photoproducts in aqueous solution at alkaline pH being the corresponding isoguanine nucleotides. The modified ring vibration pattern of the 1-oxide analogues as well as the 13C chemical shift indicate a loss of aromaticity as compared to the natural compounds. Coupling constant measurements show that the dihedral angle between the 31POC and OC13C planes is around 180degree, i.e., trans, as in the natural adenine nucleotides. The modified adenine nucleotides were tested as potential substrates and/or inhibitors of mitochondrial processes, as substrates of varous phosphotransferases from mitochondria or cytosol, and as allosteric effectors in the reactions catalyzed by glutamate dehydrogenase and phosphofructokinase. Although the adenine 1-oxide nucleotides are not recognized by the translocase system of the inner mitochondrial membrane, they are good substrates for mitochondrial phosphotransferases located in the intermembrane space. Similarly, they participate in the phosphoryl group transfer reactions catalyzed by pyruvate kinase, phosphofructokinase, and hexokinase. As allosteric effectors, the modified nucleotides are less active than the natural compounds, probably because of a lower binding capacity to the allosteric sites of the regulatory enzymes.

Participation of N1-oxide derivatives of adenine nucleotides in the phosphotransferase activity of liver mitochondria.

The modified adenine nucleotides ATP-NO, ADP-NO, and AMP-NO were tested as potential substrates and/or inhibitors of mitochondrial phosphotransferases. ADP-NO is not recognized by the translocase system located in the inner mitochondrial membrane; however, it is rapidly phosphorylated to ATP-NO in the outer compartment of mitochondria, by way of the nucleosidediphosphate kinase (EC 2.7.4.6) reaction, provided there is sufficient ATP in the mitochondria. AMP-NO is not phosphorylated by liver mitochondria to the corresponding nucleoside diphosphate; it cannot serve as substrate for adenylate kinase (EC 2.7.4.3). ATP-NO and ADP-NO, however, are substrates of this enzyme. The apparent equilibrium constant for the reaction, ADP-NO + ADP right harpoon over left harpoon ATP-NO + AMP, of 0.908 at pH 7.4 and 5 mM Mg(2+) is significantly higher than that of the reaction with natural nucleotides. Although adenosine N(1)-oxide is easily phosphorylated to AMP-NO by adenosine kinase [Schnebli et al. (1967) J. Biol. Chem. 242, 1997-2004], the formation of corresponding nucleoside triphosphate in vivo seems also to be limited by adenylate kinase; adenosine N(1)-oxide cannot replace adenosine in restoring the normal ATP level in ethionine-treated rats.