3-Hydroxykynurenine and 3-hydroxyanthranilic acid generate hydrogen peroxide and promote alpha-crystallin cross-linking by metal ion reduction.

The kynurenine pathway catabolite 3-hydroxykynurenine (3HK) and redox-active metals such as copper and iron are implicated in cataractogenesis. Here we investigate the reaction of kynurenine pathway catabolites with copper and iron, as well as interactions with the major lenticular structural proteins, the alpha-crystallins. The o-aminophenol kynurenine catabolites 3HK and 3-hydroxyanthranilic acid (3HAA) reduced Cu(II)>Fe(III) to Cu(I) and Fe(II), respectively, whereas quinolinic acid and the nonphenolic kynurenine catabolites kynurenine and anthranilic acid did not reduce either metal. Both 3HK and 3HAA generated superoxide and hydrogen peroxide in a copper-dependent manner. In addition, 3HK and 3HAA fostered copper-dependent alpha-crystallin cross-linking. 3HK- or 3HAA-modifed alpha-crystallin showed enhanced redox activity in comparison to unmodified alpha-crystallin or ascorbate-modified alpha-crystallin. These data support the possibility that 3HK and 3HAA may be cofactors in the oxidative damage of proteins, such as alpha-crystallin, through interactions with redox-active metals and especially copper. These findings may have relevance for understanding cataractogenesis and other degenerative conditions in which the kynurenine pathway is activated.

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 >> mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.

The A beta peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction.

Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid A beta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry. Spectrophotometric experiments establish that the A beta peptide reduces Fe(III) and Cu(II) to Fe(II) and Cu(I), respectively. Spectrochemical techniques are used to show that molecular oxygen is then trapped by A beta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts of Fe(II) or Cu(I). In the presence of Cu(II) or Fe(III), A beta produces a positive thiobarbituric-reactive substance (TBARS) assay, compatible with the generation of the hydroxyl radical (OH.). The amounts of both reduced metal and TBARS reactivity are greatest when generated by A beta 1-42 >> A beta 1-40 > rat A beta 1-40, a chemical relationship that correlates with the participation of the native peptides in amyloid pathology. These findings indicate that the accumulation of A beta could be a direct source of oxidative stress in Alzheimer's disease.

Protein analyst--a distributed object environment for protein sequence and structure analysis.

SUMMARY: Protein Analyst is a flexible tool for the analysis of protein sequences with emphasis on the integration of sequence and structural information. AVAILABILITY: The software will be available from the Oxford Molecular Biolib web site (http://www. oxmol.co.uk/biolib) and will be free to the academic research community.

Development and testing of the Resistiveness to Care Scale.

A conceptual model and objective scale for measuring resistiveness to care in individuals with advanced dementia of the Alzheimer type (DAT) were empirically generated from the perspective of nursing staff caregivers and through observation of residents with DAT. The resistiveness to care scale (RTC-DAT) was judged to have content validity and reduced to 13 items. Quantifiable scoring procedures and methods for rating videotapes and conducting clinical observations were developed. The RTC-DAT was tested with 68 subjects at three sites. The RTC has a range of 0-156. Initial testing provided reliability estimates of .82-.87 for internal consistency and good to excellent kappas. Criterion-related validity with observed discomfort and construct validity by factor analysis support the RTC-DAT. Measurement issues and recommendations for use in research are discussed.

Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis.

The cortical deposition of Abeta is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Abeta, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Abeta at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Abeta1-40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Abeta1-40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Abeta1-40 and Abeta1-42 with submicromolar concentrations of Cu2+. Unlike Abeta1-40, Abeta1-42 was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Abeta1-40 and histidine-modified human Abeta1-40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Abeta assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Abeta that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Abeta aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Abeta explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer's disease.

Zinc-induced Alzheimer's Abeta1-40 aggregation is mediated by conformational factors.

The heterogeneous precipitates of Abeta that accumulate in the brain cortex in Alzheimer's disease possess varying degrees of resistance to resolubilization. We previously found that Abeta1-40 is rapidly precipitated in vitro by physiological concentrations of zinc, a neurochemical that is highly abundant in brain compartments where Abeta is most likely to precipitate. We now present evidence that the zinc-induced precipitation of Abeta is mediated by a peptide dimer and favored by conditions that promote alpha-helical and diminish beta-sheet conformations. The manner in which the synthetic peptide is solubilized was critical to its behavior in vitro. Zinc-induced Abeta aggregation was dependent upon the presence of NaCl, was enhanced by alpha-helical-promoting solvents, but was abolished when the peptide stock solution was stored frozen. The Abeta aggregates induced by zinc were reversible by chelation, but could then be reprecipitated by zinc for several cycles, indicating that the peptide's conformation is probably preserved in the zinc-mediated assembly. In contrast, Abeta aggregates induced by low pH (5.5) were not resolubilized by returning the pH milieu to 7.4. The zinc-Abeta interaction exhibits features resembling the gelation process of zinc-mediated fibrin assembly, suggesting that, in events such as clot formation or injury, reversible Abeta assembly could be physiologically purposive. Such a mechanism is contemplated in the early evolution of diffuse plaques in Alzheimer's disease and suggests a possible therapeutic strategy for the resolubilization of some forms of Abeta deposit in the disease.

Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2'-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S-/(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

The solution structure of echistatin: evidence for disulphide bond rearrangement in homologous snake toxins.

The solution structure of the fibrinogen antagonist, echistatin, has been determined by a combination of NMR and simulated annealing methods. While the structure of the disulphide-linked core is well-defined by the NMR data, the N- and C-termini and the loop bearing the RGD sequence (which is responsible for the fibrinogen antagonist properties) are poorly defined. The pattern of disulphide bridges, which could not be determined by classical methods, was predicted by a statistical analysis of the simulated annealing structures. This pattern is distinct from that for the homologous protein kistrin, leading to the novel suggestion that homologous proteins possess non-conserved patterns of disulphide bridges.

Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport.

The ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization. This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export of yeast a-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR). Here we present a tertiary structure model of the ATP-binding cassettes characteristic of this class of transport system, based on similarities between the predicted secondary structures of members of this family and the previously determined structure of adenylate kinase. This model has implications for both the molecular basis of transport and cystic fibrosis and provides a framework for further experimentation.

Immunofluorescent localization of type II insulin-like growth factor receptor in rat liver and hepatoma cells.

We have used an immunofluorescent technique to localize type II insulin-like growth factor (IGF) receptors in rat liver, rat hepatocytes and three rat hepatoma cell lines (HTC, H-35 and 5123) using a polyclonal antibody (C-1) raised to purified rat liver type II IGF receptor. Specificity of the antiserum was confirmed by Western blotting of microsomal membranes prepared from hepatocytes and hepatoma cells which showed a single class of receptor in all cells, of Mr approximately 210,000 for hepatocytes, HTC and H-35 cells and approximately 220,000 for 5123 cells, on non-reduced, 4-15% polyacrylamide gradient gels. The specificity of the immunofluorescent technique was also verified by abolition of labelling after preincubation of antiserum with purified type II IGF receptor. Rat liver cryosections contained areas of juxtanuclear labelling in hepatocytes, consistent with the presence of type II IGF receptor in the Golgi region. Brightest immunofluorescence was seen in sections from fetal and neonatal rats with adult rat hepatocytes staining brightly only around central veins. Areas of labelling were also seen in connective tissue surrounding larger veins. Cultured adult rat hepatocytes and rat hepatoma cell lines also showed bright areas of juxtanuclear nuclear immunofluorescence, with HTC and H-35 cells staining more than 5123 and adult hepatocytes. Fetal rat hepatocytes in culture also labelled very brightly both in a juxtanuclear location and in small clusters over the cell, possibly on the cell surface. These observations indicate that type II IGF receptors are located predominantly on intracellular membranes and are most abundant in rapidly growing cells and tissues (such as fetal liver and hepatoma cells).

The response of megakaryocytes with processes to thrombin.

The response of megakaryocytes to thrombin (1-10 U/ml) has been examined by time-lapse cinemicrography and electron microscopy. The study was confined to mature megakaryocytes which had developed processes following incubation in vitro. The initial response of all cells was to undergo retraction of processes, behaviour thought to be linked with the depolymerization of microtubules which extend longitudinally through the processes. The majority of cells completely withdrew their processes, but about 30% responded differently and underwent only limited retraction, followed by secretion. Analysis of time-lapse film showed that processes from the latter group of cells had formed attachments with the coverslip prior to exposure to thrombin. Within the partially retracted processes of these cells, secretory granules were found to be clustered centripetally and enveloped by a microfilamentous structure in the form of a cylinder. Vacuoles appeared, some of which were located outside the microfilamentous structure. Microtubules were present, but many appeared disorientated. The shape of the microfilamentous structure suggests that the cytoplasm is not organized into putative platelets at the time of process formation.

Megakaryocyte fragments and the microtubule coil.

We have examined megakaryocyte process fragments that migrate out of bone marrow explants after a short period of incubation and assume a beaded form, consisting of 2 or more putative platelets. The fragmentation appears to occur in vivo and supports the proposal that platelet liberation does not always occur in a sequential manner from the distal ends of megakaryocyte processes. Transmission electron microscopy revealed that microtubules were generally oriented longitudinally in the process fragments. Rarely, a microtubule coil was found in a terminally located putative platelet. The observations favour the view that the marginal coil of microtubules, which is a characteristic of circulating platelets, does not usually form until after platelets have been liberated.