Functional Characterization of ECP-Heparin Interaction: A Novel Molecular Model

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]₃, and residues Gln⁴⁰, His⁶⁴ and Arg¹⁰⁵ were indicated as major contributions for the interaction. Interestingly, Gln⁴⁰ and His⁶⁴ on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln⁴⁰-His⁶⁴ clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.     

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.

Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species (∼10–15 million years ago). Nonsynonymous substitutions per site (d _N) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (d _N / d _S) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense. Received: 8 April 1999 / Accepted: 22 June 1999     

Triazole double-headed ribonucleosides as inhibitors of eosinophil derived neurotoxin

Eosinophil derived neurotoxin (EDN) is an eosinophil secretion protein and a member of the Ribonuclease A (RNase A) superfamily involved in the immune response system and inflammatory disorders. The pathological actions of EDN are strongly dependent on the enzymatic activity and therefore, it is of significant interest to discover potent and specific inhibitors of EDN. In this framework we have assessed the inhibitory potency of triazole double-headed ribonucleosides. We present here an efficient method for the heterologous production and purification of EDN together with the synthesis of nucleosides and their biochemical evaluation in RNase A and EDN. Two groups of double-headed nucleosides were synthesized by the attachment of a purine or a pyrimidine base, through a triazole group at the 3′-C position of a pyrimidine or a purine ribonucleoside, respectively. Based on previous data with mononucleosides these compounds were expected to improve the inhibitory potency for RNase A and specificity for EDN. Kinetics data revealed that despite the rational, all but one, double-headed ribonucleosides were less potent than the respective mononucleosides while they were also more specific for ribonuclease A than for EDN. Compound 11c (9-[3′-[4-[(cytosine-1-yl)methyl]-1,2,3-triazol-1-yl]-β-d-ribofuranosyl]adenine) displayed a stronger preference for EDN than for ribonuclease A and a K_i value of 58 μM. This is the first time that an inhibitor is reported to have a better potency for EDN than for RNase A. The crystal structure of EDN–11c complex reveals the structural basis of its potency and selectivity providing important guidelines for future structure-based inhibitor design efforts.     

Rational immunotherapy with ribonuclease chimeras

Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC₅₀ around 10⁻⁷ M, whereas>10⁻⁴ M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab′)₂-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1–5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.     

The Correlation of RNase A Enzymatic Activity with the Changes in the Distance between Nε2-His12 and Nδ1-His119 Upon Addition of Stabilizing and Destabilizing Salts

The effect of stabilizing and destabilizing salts on the catalytic behavior of ribonuclease A (RNase A) was investigated at pH 7.5 and 25°C, using spectrophotometric, viscometric and molecular dynamic methods. The changes in the distance between N_ε2 of His₁₂ and N_δ1 of His₁₁₉ at the catalytic center of RNase A upon the addition of sodium sulfate, sodium hydrogen sulfate and sodium thiocyanate were evaluated by molecular dynamic methods. The compactness and expansion in terms of Stokes radius of RNase A upon the addition of sulfate ions as kosmotropic salts, and thiocyanate ion as a chaotropic salt, were estimated by viscometric measurements. Enzyme activity was measured using cytidine 2′, 3′-cyclic monophosphate as a substrate. The results from the measurements of distances between N_ε2 of His₁₂ and N_δ1 of His₁₁₉ and Stokes radius suggest (i) that the presence of sulfate ions decreases the distance between the catalytic His residues and increases the globular compactness, and (ii) that there is an expansion of the enzyme surface as well as elongation of the catalytic center in the presence of thiocyanate ion. These findings are in agreement with activity measurements.     

Network of long-range concerted chemical shift displacements upon ligand binding to human angiogenin

Molecular recognition models of both induced fit and conformational selection rely on coupled networks of flexible residues and/or structural rearrangements to promote protein function. While the atomic details of these motional events still remain elusive, members of the pancreatic ribonuclease superfamily were previously shown to depend on subtle conformational heterogeneity for optimal catalytic function. Human angiogenin, a structural homologue of bovine pancreatic RNase A, induces blood vessel formation and relies on a weak yet functionally mandatory ribonucleolytic activity to promote neovascularization. Here, we use the NMR chemical shift projection analysis (CHESPA) to clarify the mechanism of ligand binding in human angiogenin, further providing information on long-range intramolecular residue networks potentially involved in the function of this enzyme. We identify two main clusters of residue networks displaying correlated linear chemical shift trajectories upon binding of substrate fragments to the purine- and pyrimidine-specific subsites of the catalytic cleft. A large correlated residue network clusters in the region corresponding to the V₁ domain, a site generally associated with the angiogenic response and structural stability of the enzyme. Another correlated network (residues 40–42) negatively affects the catalytic activity but also increases the angiogenic activity. ¹⁵N-CPMG relaxation dispersion experiments could not reveal the existence of millisecond timescale conformational exchange in this enzyme, a lack of flexibility supported by the very low-binding affinities and catalytic activity of angiogenin. Altogether, the current report potentially highlights the existence of long-range dynamic reorganization of the structure upon distinct subsite binding events in human angiogenin.     

Sequence-specific backbone resonance assignments and microsecond timescale molecular dynamics simulation of human eosinophil-derived neurotoxin

Eight active canonical members of the pancreatic-like ribonuclease A (RNase A) superfamily have been identified in human. All structural homologs share similar RNA-degrading functions, while also cumulating other various biological activities in different tissues. The functional homologs eosinophil-derived neurotoxin (EDN, or RNase 2) and eosinophil cationic protein (ECP, or RNase 3) are known to be expressed and secreted by eosinophils in response to infection, and have thus been postulated to play an important role in host defense and inflammatory response. We recently initiated the biophysical and dynamical investigation of several vertebrate RNase homologs and observed that clustering residue dynamics appear to be linked with the phylogeny and biological specificity of several members. Here we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments of human EDN (RNase 2) and its molecular dynamics simulation on the microsecond timescale, providing means to pursue this comparative atomic-scale functional and dynamical analysis by NMR and computation over multiple time frames.     

Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein

The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.     

Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction

Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.     

Ribonuclease production of Rhizopus oryzae IFO 4697 under metal ion stress in liquid culture

 Rhizopus oryzae IFO 4697 was found to produce intracellular ribonuclease (RNase), and its growth and activity could be regulated under selected metal ion stress. The addition of Fe²⁺, Mg²⁺ and Zn²⁺ to the SLSR medium was essential to growth and RNase production. Ca²⁺ and Mo⁶⁺ stimulated RNase production. It is concluded that the addition of 100 mg/ml Ca²⁺, 5 mg/ml Mo⁶⁺, 0.7 mg/ml Zn²⁺, 2 mg/ml Fe²⁺, and 49 mg/ml Mg²⁺ to the SLSR medium was the best condition for producing RNase in high specific activity (3780 U/mg protein). This result indicates that a metal ion-regulated liquid medium is an efficient culture method for RNase production. Received: July 19, 2001 / Accepted: April 8, 2002     

Recognition of ribonuclease A by 3'-5'-pyrophosphate-linked dinucleotide inhibitors: a molecular dynamics/continuum electrostatics analysis

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5′-phospho-2′-deoxyuridine-3-pyrophosphate (P→5)-adenosine-3-phosphate (pdUppA-3′-p). The 3′,5′-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B₂ and the placement of the 5′-β-phosphate of the adenylate (instead of the α-phosphate) at subsite P₁ where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3′-p and the related weaker inhibitor dUppA, which lacks the 3′ and 5′ terminal phosphate groups of pdUppA-3′-p. The simulations show that the adenylate 5′-β-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P₁ and B₂. The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3′-p varies between −7.9 kcal/mol and −2.8 kcal/mol for a range of protein/ligand dielectric constants (ε_p) 2–20, in good agreement with the experimental value (−3.6 kcal/mol); the agreement becomes exact with ε_p = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3′-p make increased interactions with residues Lys⁷ and Lys⁶⁶ of the more remote sites P₂ and P₀, and His¹¹⁹ of site P₁.     

RNase 8, a novel RNase A superfamily ribonuclease expressed uniquely in placenta

We report the identification and characterization of the gene encoding the eighth and final human ribonuclease (RNase) of the highly diversified RNase A superfamily. The RNase 8 gene is linked to seven other RNase A superfamily genes on chromosome 14. It is expressed prominently in the placenta, but is not detected in any other tissues examined. Phylogenetic analysis suggests that RNase 7 is the closest relative of RNase 8 and that the pair likely resulted from a recent gene duplication event in primates. Further analysis reveals that the RNase 8 gene has incorporated non-silent mutations at an elevated rate (1.3 × 10^–9 substitutions/site/year) and that orthologous RNase 8 genes from 6 of 10 primate species examined have been deactivated by frameshifting deletions or point mutations at crucial structural or catalytic residues. The ribonucleolytic activity of recombinant human RNase 8 is among the lowest of members of this superfamily and it exhibits neither antiviral nor antibacterial activities characteristic of some other RNase A ribonucleases. The rapid evolution, species-limited deactivation and tissue-specific expression of RNase 8 suggest a unique physiological function and reiterates the evolutionary plasticity of the RNase A superfamily.     

Chromosomal assignment of a large tRNA gene cluster (tRNALeu,tRNAGln, tRNALys,tRNAArg, tRNAGly) to 17p13.1

Summary A cluster of tRNA genes (tRNA _UAG ^Leu , tRNA _CUG ^Gln , tRNA _UUU ^Lys , tRNA _UCU ^Arg ) and an adjacent tRNA _GCC ^Gly have been assigned to human chromosome 17p12–p13.1 by in situ hybridization using a 4.2 kb human DNA fragment for tRNA^Leu, tRNA^Gln, tRNA^Lys, tRNA^Arg, and, for tRNA^Gly, 1.3 kb and 0.58 kb human DNA fragments containing these genes as probes. This localization was confirmed and refined to 17p13.100–p13.105 using a somatic cell hybrid mapping panel. Preliminary experiments with the biotiny lated tRNA Leu, Gln, Lys, Arg probe and metaphase spreads from other great apes suggest the presence of a hybridization site on the long arm of gorilla (Gorilla gorilla) chromosome 19 and the short arm of orangutan (Pongo pygmaeus) chromosome 19 providing further support for homology between HSA17, GGO19 and PPY19.     

The role of microbial dormancy autoinducers in metabolism blockade

Alkyl-substituted hydroxybenzenes (AHBs), which are autoinducers of microbial dormancy (d ₁ factors), were found to stabilize the structure of protein macromolecules and modify the catalytic activity of enzymes. In vitro experiments showed that C₆-AHB at concentrations from 10⁻⁴ to 10⁻² M, at which it occurs in the medium as a true solution and a micellar colloid, respectively, nonspecifically inhibited the activity of chymotrypsin, RNase, invertase, and glucose oxidase. C⁶-AHB-induced conformational alterations in protein macromolecules were due to the formation of complexes, as evidenced by differences in the fluorescence spectra of individual RNase and C₆-AHB and their mixtures and in the surface tension isotherms of C₆-AHB and trypsin solutions. Data on the involvement of dormancy autoinducers in the posttranslational modification of enzymes and their inhibition will provide further insight into the mechanisms of development and maintenance of dormant microbial forms.     

A Sequential Mechanism for Exosite-mediated Factor IX Activation by Factor XIa

During blood coagulation, the protease factor XIa (fXIa) activates factor IX (fIX). We describe a new mechanism for this process. FIX is cleaved initially after Arg¹⁴⁵ to form fIXα, and then after Arg¹⁸⁰ to form the protease fIXaβ. FIXα is released from fXIa, and must rebind for cleavage after Arg¹⁸⁰ to occur. Catalytic efficiency of cleavage after Arg¹⁸⁰ is 7-fold greater than for cleavage after Arg¹⁴⁵, limiting fIXα accumulation. FXIa contains four apple domains (A1–A4) and a catalytic domain. Exosite(s) on fXIa are required for fIX binding, however, there is lack of consensus on their location(s), with sites on the A2, A3, and catalytic domains described. Replacing the A3 domain with the prekallikrein A3 domain increases K_m for fIX cleavage after Arg¹⁴⁵ and Arg¹⁸⁰ 25- and ≥90-fold, respectively, and markedly decreases k_cat for cleavage after Arg¹⁸⁰. Similar results were obtained with the isolated fXIa catalytic domain, or fXIa in the absence of Ca²⁺. Forms of fXIa lacking the A3 domain exhibit 15-fold lower catalytic efficiency for cleavage after Arg¹⁸⁰ than for cleavage after Arg¹⁴⁵, resulting in fIXα accumulation. Replacing the A2 domain does not affect fIX activation. The results demonstrate that fXIa activates fIX by an exosite- and Ca²⁺-mediated release-rebind mechanism in which efficiency of the second cleavage is enhanced by conformational changes resulting from the first cleavage. Initial binding of fIX and fIXα requires an exosite on the fXIa A3 domain, but not the A2 or catalytic domain.     

Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity.

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.     

A unique β-agarase,AgaA, from a marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. strain PO-303

The agaA gene encoding β-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40°C and pH 7.5, with a specific activity of 16.4 units mg⁻¹, a K _m of 1.10 mg ml⁻¹, and a V _max of 22.5 μmol min⁻¹ mg⁻¹ for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.     

DNA repair XRCC1 Arg399Gln polymorphism is associated with the risk of development of end-stage renal disease

Patients with end-stage renal disease (ESRD) display enhanced genomic damage. DNA repair gene polymorphisms may affect DNA repair capacity and modulate susceptibility to ESRD. In this study, we aimed to determine the frequency of polymorphisms in two DNA repair enzyme genes, Xeroderma pigmentosum complementation group D (XPD) and X-ray cross-complementing group 1 (XRCC1), in patients with ESRD and to evaluate their association with ESRD development. By using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), we genotyped four single nucleotide polymorphisms (SNPs) in XPD codons 312 and 751 and XRCC1 codons 194 and 399 in 136 dialysis patients (71 patients undergoing hemodialysis and 65 subjected to peritoneal dialysis) and 147 healthy controls. Patients having XRCC1 399 Arg/Gln (OR:1.98; 95% CI: 1.21–3.25, P = 0.007) or XRCC1-399 Gln/Gln (OR: 3.95; 95% CI: 1.45–10.76, P = 0.005) genotype had a significantly higher risk of ESRD than those with XRCC1 399 Arg/Arg genotype. We also found a significantly higher frequency of the XRCC1 399Gln allele in patients with ESRD than in controls, with OR = 2.03 (95% CI = 1.08–3.81, P = 0.03). We further investigated the potential combined effect of these DNA repair variants on the risk of ESRD development. It was found that combination of the Arg/Gln or Gln/Gln genotypes of XRCC1 Arg399Gln polymorphism with the two possible genotypes of XPD-Asp312Asn or with the Lys/Gln or Gln/Gln genotypes of XPD Lys751Gln was significantly associated with the development of ESRD. This is the first report showing an association between DNA repair gene polymorphisms and ESRD development, and suggests that XRCC1 Arg399Gln polymorphism may confer increased risk for the development of the disease. Further larger studies should be conducted to confirm these results.     

Performance of the isolated rainbow trout heart perfused under self-controlled coronary pressure conditions: effects of high and low oxygen tension, arachidonic acid and indomethacin

The effects of arachidonic acid (AA) and indomethacin (IM) on performance, oxygen consumption and lactate release of the trout heart were studied in vitro TPa s m⁻³ using a perfusion system, which allowed the evaluation of the integrated function of ventricle and coronary system by continuously setting the input coronary flow and pressure proportional to the pressure and flow output of the heart. The heart was working against a fixed resistance. A reduction of input oxygen partial pressure (P_O2) from 175 torr (high P_O2) to 76 torr (low P_O2) increased the coronary flow (from 0.51 ml min⁻¹ kg⁻¹ to 1.21 ml min⁻¹ kg⁻¹, respectively) due to a strong reduction in coronary resistance (from 0.60 TPa s m⁻³ to 0.19 TPa s m⁻³, respectively). Oxygen consumption by the heart was significantly reduced from 20.7 ml min⁻¹ g⁻¹ at high P_O2 to 4.6 ml min⁻¹ g⁻¹ at low P_O2, while lactate production was increased from 24 μmol h⁻¹ g⁻¹ to 42 μmol h⁻¹ g⁻¹, indicating a higher contribution of anaerobic respiration to mechanical work. Mechanical efficiency was significantly higher at low than at high P_O2. Exogenous AA caused a depression of inotropism and a reduction in the aerobic metabolic rate (by 25–35%), which was not accompanied by increased lactate production. IM enhanced the depression of both inotropism and aerobic metabolism. The effect of AA and IM on the heart were amplified at low P_O2. Accepted: 20 October 1997