Large neutral amino acids (LNAAs) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In earlier studies on mice with PKU (ENU(2)/ENU(2)), LNAAs were given and a surprising decline in blood Phe concentrations was observed. The formula used in the mouse experiment (PreKUnil) lacked lysine. Therefore, a new formulation of LNAAs (NeoPhe) was developed, introducing changes in the concentration of some amino acids and adding lysine, so that such a mixture could be used in humans. The new formula was found to be effective in reducing blood Phe concentration in mice by about 50% of the elevated levels. Patients with PKU were given LNAAs and blood Phe concentrations were determined in an open-label study. Three centers--in Russia, the Ukraine and the USA--took part in the study. NeoPhe was given at 0.5 g/kg per day in three divided doses to eight subjects with PKU and at 1.0 g/kg per day to three patients, for one week. The NeoPhe resulted in decrease of elevated blood Phe by 50% in both groups. The preliminary data from this study are encouraging and a double blind placebo-controlled trial will be required to show long-term efficacy and tolerance of LNAAs in the treatment of PKU.

Large neutral amino acids (LNAA) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In an open-label study using LNAA, a surprising decline of blood Phe concentration was found in patients with PKU in metabolic treatment centres in Russia, the Ukraine, and the United States. To validate the data obtained from this trial, a short-term double-blind placebo control study was done using LNAA in patients with PKU, with the participation of three additional metabolic centres--Milan, Padua and Rio de Janeiro. The results of the short trial showed significant lowering of blood Phe concentration by an average of 39% from baseline. The data from the double-blind placebo control are encouraging, establishing proof of principle of the role of orally administered LNAA in lowering blood Phe concentrations in patients with PKU. Long-term studies will be needed to validate the acceptability, efficacy and safety of such treatment.

We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314 -28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and K m for this reaction was ϳ200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.

Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5′ base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Polκ and Polζ function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Polκ at the insertion step and of Polζ at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.DNA polymerases kappa and zeta | error-free bypass of thymine glycol | replicative lesion bypass | thymine glycol bypass in humans T hymine glycol (Tg) is the most common oxidation product of thymine (1-3). Hydroxyl radicals, the principal DNA damaging species formed from aerobic respiration or from exposure to chemical oxidizing agents or ionizing radiation, interact directly with thymine primarily at the 5,6 double bond, generating a thymine glycol. Although Tg lesions in humans can be removed by base excision repair (BER) involving the action of the DNA glycosylase/AP lyase NTH1 or by nucleotide excision repair (NER) (4, 5), if the damaged base is not removed by these repair processes and goes through the replication fork, that presents a severe block to continued synthesis by eukaryotic replicative DNA polymerases (Pols) (6, 7).Because of the addition of hydroxyl groups at C5 and C6 in a Tg, the damaged base loses its aromatic character and becomes nonplanar (8-10). As a consequence, the C5 methyl group protrudes in an axial direction that prevents the base 5′ to the Tg lesion from stacking above it. Hence a Tg lesion presents a block not only for nucleotide (nt) insertion opposite it but also for the subsequent extension step. Steady-state kinetic analyses of Tg bypass by human translesion (TLS) Pols have indicated that Polη can insert an A opposite Tg with the same catalytic efficiency as for insertion of an A opposite undamaged T; moreover, Polη can also extend from the Tg:A base pair with only an ∼15-fold reduction in efficiency (7). However, Polη incorporates the wrong nts opposite Tg rather frequently as the misincorporation of G or a C residue occurs with only an ∼8-, and 20-fold reduction in effici...

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421-431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421-436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421-436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.Many enzymes exploit covalent interactions with substrates to catalyze chemical transformations. On the other hand, most studies on Ab 1 catalysis have focused on non-covalent binding forces as the mechanism by which the energy barrier between reactants and products is lowered, e.g. the electrostatic forces that stabilize the negatively charged oxyanionic transition state of ester hydrolysis (reviewed in Refs. 1 and 2). The underlying assumption has been that Abs interact with their ligands exclusively by non-covalent means. Initial indications that natural Abs express chemical reactivity indistinguishable from enzymes came from reports of proteolytic and nuclease activity of autoantibodies (3, 4). Similar activities were later found in Ab light chains from multiple myeloma patients (5), alloantibodies from patients with transfusion-induced hemophilia (6), Abs raised by routine immunization with polypeptides (7, 8), and anti-idiotypic Abs to anti-enzyme Abs (9). From mutagenesis and inhibitor studies, it appears that the proteolytic activity of natural Abs originates from nucleophilic mechanisms similar to those utilized by conventional serine proteases (10, 11). The catalytic activity of natural Abs could be construed to violate the pri...

A favorable response, indicated by decline of blood phenylalanine (Phe) in patients with phenylketonuria (PKU), to orally administered 6-R-L-erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4) has been reported in many countries following the first publication in 1999. In this review, we describe the experience in the United States with PKU patients and their response to BH4. A significant response to BH4 is arbitrarily considered as a decrease of 30% or greater of blood Phe concentration 24 h after administration of BH4. In our studies, 18 of 37 patients with PKU (49%) responded to oral BH4 by >30% decrease in blood Phe concentration. Four PKU patients responded with a decrease of blood Phe concentration between 17.3 and 26.3%. It is suggested that patients with sufficient response to BH4 are candidates who will benefit from BH4 as it becomes available for PKU management. In a separate trial, 20 patients with PKU were screened with ascending doses of BH4: 10, 20, and 40 mg/kg. A favorable response was found in 10 subjects (50%) after 10 mg/kg BH4 and 14 subjects (70%) after 20 mg/kg BH4. There was no additional advantage to 40 mg/kg BH4. A 1-wk trial with 10 and 20 mg/kg BH4 in the same 20 patients showed blood Phe concentrations lowest after 7 d of BH4. The BH4-responsive patients were genotyped and most were compound heterozygotes with 1 mild mutation on 1 allele, responsible for the increase of the residual activity of Phe hydroxylase when BH4 was added. Individuals with the same genotype exhibit different responses upon administration of BH4, attributed to epigenetic factors, such as the metabolic makeup of the individual. Patients with PKU, regardless of their genotype or classification, need to be screened for response to BH4. The majority of patients are identified by 10 mg/kg BH4.

The growing use of N-acetylaspartate as an indicator of neuronal viability has fostered interest in the biological function(s) of this unusual amino acid derivative. In considering the various physiological roles that have been proposed for this relatively abundant molecule one is obliged to take into account its unusual metabolic compartmentalization, according to which synthesis and storage occur in the neuron and hydrolytic cleavage in the oligodendrocyte. The latter reaction, catalyzed by aspartoacylase (ASPA), produces acetyl groups plus aspartate and has been proposed to occur in both soluble and membranous subfractions of white matter. Our study supports such bimodal occurrence and we now present immunoblot, proteomic, and biochemical evidence that the membrane-bound form of ASPA is intrinsic to purified myelin membranes. This was supported by a novel TLC-based method for the assay of ASPA. That observation, together with previous demonstrations of numerous lipid-synthesizing enzymes in myelin, suggests utilization of acetyl groups liberated by myelin-localized ASPA for lipid synthesis within the myelin sheath. Such synthesis might be selective and could explain the deficit of myelin lipids in animals lacking ASPA.

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