Increased levels of pregnancy-associated plasma protein A are associated with mortality in hemodialysis patients: preliminary results.

Pregnancy-associated plasma protein A (PAPP-A) is a new prognostic indicator of acute coronary syndrome. This protein is elevated in hemodialysis (HD) patients and is closely related to inflammation and oxidative stress. The aim of our pilot study was to find out whether PAPP-A is related to mortality in HD patients. 40 HD patients in a stable clinical state (20 men and 20 women, mean age 69 +/- 12 years) were enrolled in the study and followed up for 20 months. PAPP-A was assessed immunochemically (TRACE method) in serum samples (before the HD session) at the beginning of the observation period. During the follow-up, 22 patients died, 15 of them due to cardiovascular events. PAPP-A levels were significantly higher in the patients who died, compared to living HD patients: 26.8 (21.6-36.8) vs. 20 (14.9-26.6) mU/l, p = 0.034. PAPP-A could also be a new prognostic marker in hemodialysis patients, probably due to its close association with cardiovascular risk. More extensive studies are required to confirm this hypothesis.

Relationship of pregnancy-associated plasma protein-a to renal function and dialysis modalities.

BACKGROUND: The aim of the study was to determine pregnancy-associated plasma protein-A (PAPP-A), which was recently described as a new marker of cardiovascular events, in patients with chronic renal insufficiency/failure and to find out its relationship to renal function and to prominent markers of oxidative stress (advanced oxidation protein products--AOPP) and inflammation (C-reactive protein--CRP). METHODS: The studied group consisted of 36 chronic hemodialysis patients (HD), 10 patients treated with continuous ambulatory peritoneal dialysis (CAPD) and 38 patients with chronic renal insufficiency (CHRI) not yet dialyzed. PAPP-A was measured by Time Resolved Amplified Cryptate Emission technology. Determination of AOPP is based on a spectrophotometric method. RESULTS: PAPP-A levels are statistically significantly elevated in the both groups of dialyzed patients in comparison with healthy subjects (27.0 +/- 16.5 mIU/l in HD and 14.07 +/- 6.73 mIU/l in CAPD vs. 8.22 +/- 2.7 mIU/l in the control group, p < 0.0001 and p < 0.001, respectively, p < 0.05 HD vs. CAPD). The mean serum PAPP-A levels in the CHRI patients not yet dialyzed were not significantly higher in comparison with the control group (9.72 +/-4.44 vs. 8.22 +/- 2.7 mIU/l, n.s.). In the CHRI not dialyzed patients, we found a significant positive correlation between serum creatinine and PAPP-A levels (r = 0.68, p < 0.05). In comparison with controls, AOPP and CRP levels were significantly higher in HD patients [AOPP 155.0 +/- 37.9 micromol/l, p < 0.0001 vs. controls, CRP 10.0 (4.6- 26.9) mg/l (median, interquartile range), p < 0.0001 vs. controls], CAPD patients [AOPP 118.5 +/- 25.8 micromol/l, p < 0.0001 vs. controls, CRP 7.7 (2.0-18.8) mg/l, p < 0.01 vs. controls] and AOPP levels in chronic renal failure patients not yet dialyzed (98.5 +/- 43.24 micromol/l, p < 0.01 vs. controls). The correlations between PAPP-A and AOPP (r = 0.49, p < 0.05) and PAPP-A and CRP (r = 0.48, p < 0.05) serum concentration were statistically significant in HD patients. In CAPD patients, neither a correlation between PAPP-A and AOPP nor a correlation between PAPP-A and CRP were found. CONCLUSION: We can conclude that serum PAPP-A levels sensitively reflect the changes in renal function, depend on dialysis modality, and may represent a novel marker associated with inflammation and oxidative stress in chronic renal failure patients.

Inhibition of HIV protease by monoclonal antibodies.

The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV-1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies.

Structural basis of HIV-1 and HIV-2 protease inhibition by a monoclonal antibody.

BACKGROUND: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS: We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.

Cleavage of vimentin by different retroviral proteases.

Proteases (PRs) of retroviruses cleave viral polyproteins into their mature structural proteins and replication enzymes. Besides this essential role in the replication cycle of retroviruses, PRs also cleave a variety of host cell proteins. We have analyzed the in vitro cleavage of mouse vimentin by proteases of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (M-PMV), myeloblastosis-associated virus (MAV), and two active-site mutants of MAV PR. Retroviral proteases display significant differences in specificity requirements. Here, we show a comparison of substrate specificities of several retroviral proteases on vimentin as a substrate. Vimentin was cleaved by all the proteases at different sites and with different rates. The results show that the physiologically important cellular protein vimentin can be degraded by different retroviral proteases.

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody.

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.

Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody.

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.

Preliminary crystallographic studies of an anti-HIV-1 protease antibody that inhibits enzyme activity.

F11.2.32, a monoclonal antibody directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross-reacts with peptides 36-46 and 36-57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36-46 and 36-57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.

The Gag-Pol encoded proteinase of an avian retrovirus expressed in E. coli can produce a novel proteinase (PR + IleGly) that is two amino acids larger at its carboxy-terminal region than the major Gag proteinase (PR).

Gag-Pol frameshift translational products of avian retroviruses (e.g., myeloblastosis associated virus, MAV) contain a putative proteinase species of 131 amino acids that maps between the NC/PR and the PR/RT processing sites. Expression in Escherichia coli of an in-frame PR precursor that contains the natural NC/PR processing site and is translationally terminated at the PR/RT site leads to formation of a Gag-Pol proteinase of the expected molecular size (131 amino acids) and a novel PR product of 126 amino acids. This product extends 2 amino acids downstream of the gag-encoded 124 amino acids, and its proteolytic cleavage is promoted by conditions favorable for enzyme catalysis, is blocked by a specific MAV proteinase inhibitor, and can be demonstrated also for corresponding peptide substrates. The new self-processing cleavage product is termed PR(+IleGly) and exhibits similar, but slower, catalytic parameters than those of the Gag PR.

Myeloblastosis associated virus (MAV) proteinase site-mutated to be HIV-like has a higher activity and allows production of infectious but morphologically altered virus.

We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.

An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase.

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV-1 PR. This dramatic alteration of MAV PR activity achieved by site-directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases.

High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli.

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

Effect of some chelating agents on the biliary excretion of mercury. 2. Relationship between the excretion of mercury and its binding to bile fractions.

Mercury binding to bile components and the correlation between the amount of mercury bound in the bile fraction 2 and the rate of mercury biliary excretion were studied in female rats exposed to intravenously injected HgCl2 after pretreatment with a series of 14 chemical agents. After pretreatment with the tested agent, 203Hg was detectable both in the bile fraction 1 and 2. Distribution pattern of 203Hg between the two fractions appeared to be linked with the chemical structure of the formed mercury complex. Pretreatment with these agents did not inhibit the formation of the bile fraction 3. By their influence on the 203Hg distribution between the bile fractions 1 and 2, the tested agents can be roughly divided into 3 groups: the content of 203Hg in the bile fraction 2 is about 10--20% and does not change significantly within the first 24 hours after 203 HgCl2 injection (cysteine, penicillamine, disodium ethylenediaminotetraacetate -- Na2EDTA, sodium diethyldithiocarbamate, sodium alanindithiocarbamate, acrylonitrile); the the 203Hg content in the bile fraction 2 increases (thiophenolacetate); the content of 203Hg in fraction 2 is initially several times higher than that in the bile fraction 1, but then decreases during the first 24 hours (2,3-dimercaptopropanol -- BAL, sodium 2,3-dimercaptopropanesulphonate, spironolactone, Thiomestron). The rate of mercury biliary excretion (Rb) was found to be closely correlated with the relative amount of mercury present in the bile fraction 2 (a2), if a2 > 30%, both in vivo (Rb = 1.077 a2 + 0.758) and invitro (Rb = 1.067 a2 + 0.519) experiments. Practically identical values of the constant accompanying a2 in the two equations seem to indicate that one of the decisive factors influencing the rate of mercury biliary excretion in rats is rather the mercury affinity for the bile fraction 2 components than the agent-induced mercury transport mechanisms. For a2 < 30% the correlation is non-linear and the excretion is rather inhibited than enhanced.