Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 antigens.     

Habitat deterioration promotes the evolution of direct development in metamorphosing species

Although metamorphosis is widespread in the animal kingdom, several species have evolved life-cycle modifications to avoid complete metamorphosis. Some species, for example, many salamanders and newts, have deleted the adult stage via a process called paedomorphosis. Others, for example, some frog species and marine invertebrates, no longer have a distinct larval stage and reach maturation via direct development. Here we study which ecological conditions can lead to the loss of metamorphosis via the evolution of direct development. To do so, we use size-structured consumer-resource models in conjunction with the adaptive-dynamics approach. In case the larval habitat deteriorates, individuals will produce larger offspring and in concert accelerate metamorphosis. Although this leads to the evolutionary transition from metamorphosis to direct development when the adult habitat is highly favorable, the population will go extinct in case the adult habitat does not provide sufficient food to escape metamorphosis. With a phylogenetic approach we furthermore show that among amphibians the transition of metamorphosis to direct development is indeed, in line with model predictions, conditional on and preceded by the evolution of larger egg sizes.     

Characterisation of dissolved organic matter in Parisian urban aquatic systems: predominance of hydrophilic and proteinaceous structures

Understanding the nature of organic matter is a necessary first step in assessing contaminant bioavailability and allowing water supply managers to optimise the treatment train in the aim of providing safe and inexpensive drinking water. This study provides further insight into the composition, structure and functional groups of dissolved organic matter (DOM) (both hydrophobic and hydrophilic) from urban aquatic systems by means of various analytical techniques (DAX-8/XAD-4 fractionation, elemental analysis, UV and FTIR spectroscopies, ¹³C and ¹⁵N isotopic analysis, size exclusion chromatography and Pyrolysis-GC-MS). The analytical range chosen for this study constitutes a powerful tool in the characterisation of DOM in urban water. The inclusion of information from one technique to the next might not only serve as a support to each one, but also as a complement. The DOM fraction from treated effluent and, more generally, DOM from urban water (i.e. receiving treated effluent) display a strong hydrophilic characteristic [i.e. low humic substance (HS) content, low SUVA], along with a high distribution in molecular weights observed by SEC and low average molecular weight. Due to the origin of this DOM, proteinaceous structures constitute the main compounds, as observed by FTIR and Py-GC-MS. Such characteristics (i.e. heterogeneity, low average molecular weight and diverse functional groups, which make up a total of N) could explain that DOM from treated effluent displayed a strong reactive potential metals pollutants as previously demonstrated.     

Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549

Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves-tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K⁺ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K⁺) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K⁺ channels are activated following reoxygenation but not during the hypoxia phase. The K⁺ currents decayed with time following reoxygenation. The decay characteristics of the K⁺ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K⁺ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K⁺ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K⁺ currents. © 1993 Wiley-Liss, Inc.     

Deliberately infecting healthy volunteers with malaria parasites: Perceptions and experiences of participants and other stakeholders in a Kenyan-based malaria infection study

Controlled human malaria infection (CHMI) studies involve the deliberate infection of healthy volunteers with malaria parasites under controlled conditions to study immune responses and/or test drug or vaccine efficacy. An empirical ethics study was embedded in a CHMI study at a Kenyan research programme to explore stakeholders’ perceptions and experiences of deliberate infection and moral implications of these. Data for this qualitative study were collected through focus group discussions, in-depth interviews and non-participant observation. Sixty-nine participants were involved, including CHMI study volunteers, community representatives and research staff. Data were managed using QSR Nvivo 10 and analysed using an inductive-deductive approach, guided by ethics literature. CHMI volunteers had reasonable understanding of the study procedures. Decisions to join were influenced by study incentives, trust in the research institution, their assessment of associated burdens and motivation to support malaria vaccine development. However, deliberate malaria infection was a highly unusual research strategy for volunteers, community representatives and some study staff. Volunteers’ experiences of physical, emotional and social burdens or harms were often greater than anticipated initially, and fluctuated over time, related to specific procedures and events. Although unlikely to deter volunteers' participation in similar studies in furture, we argue that the dissonance between level of understanding of the burdens involved and actual experiences are morally relevant in relation to community engagement, informed consent processes, and ongoing support for volunteers and research staff. We further argue that ethics oversight of CHMI studies should take account of these issues in deciding whether consent, engagement and the balance of benefits and harms are reasonable in a given context.     

“Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints

The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.     

A membrane-associated form of C-reactive protein is the galactose-specific particle receptor on rat liver macrophages

Rat liver macrophages express a galactose-specific receptor which mediates endocytosis of particles or neuraminidase-treated blood cells. From rat serum we now have isolated and purified a galactose-specific lectin by affinity chromatography. Comparative analysis of this serum galactose-binding protein with the galactose-particle receptor protein purified from rat liver macrophages and with C-reactive protein (CRP) reveals close relation or identity of these proteins. An apparent m.w. of 30,000 was determined for all three proteins by SDS-PAGE under reducing conditions and m.w. of about 130,000 by native PAGE. All three proteins exhibit the same pentameric, ring-shaped structure in electron microscopy after negative staining. Antibodies raised against the serum galactose-binding protein or against the macrophage receptor cross-react. A mAb specific for rat neo-CRP labels liver macrophages but not hepatocytes and reacts with the isolated protein in a Western blot assay. Furthermore, the galactose-particle receptor can be functionally replaced by purified CRP: the binding capacity for neuraminidase-treated E of receptor-depleted liver macrophages can be restored by preincubation with purified rat CRP. We therefore conclude that CRP occurs as a membrane-associated protein constitutively expressed on liver macrophages functioning as a receptor mediating galactose-specific binding of particulate ligands.     

Monoclonal antibody recognizes a conformational epitope in a random coil protein

The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not recognize intact native cytochrome c, but they crossreact with the heme-containing peptides 1-38 and 1-65 of cytochrome c. The antigenic determinant recognized by monoclonal antibody SJL 2-4 is conformational and discontiguous, it is composed of residues close to the N-terminus and around position 25. The other monoclonal antibody, Cyt-1-59, seems to recognize a contiguous epitope close to the N-terminus. The present results show that even a seemingly disordered protein which is conventionally classified as a random coil may feature subtle spatial regularities. The presence of ordered conformational elements in apocytochrome c may be important for the enzyme-catalyzed covalent attachment of the heme and the import of cytochrome c into mitochondria. A discontiguous determinant for SJL 2-4 is particularly interesting because this antibody inhibits the proliferation of a T-cell clone specific for apo-cytochrome c [Corradin & Engers (1984) Nature (Lond.) 308, 547-548].     

The binding of binuclear platinum(II)-terpyridine complexes to DNA.

The binding of platinum (II)-terpyridine complexes to DNA was studied by using equilibrium dialysis. Optical absorption methods were used to measure the ability of the ligands to aggregate in aqueous buffer. Scatchard plots for the binding of the monomeric [Pt(terpy)SC4H9]+ cation to DNA at I0.01 are curvilinear, concave upwards, suggesting two modes of binding. The association constant decreases at higher ionic strengths, consistent with polyelectrolyte theory, and 1.1 cations are released per bound ligand molecule. The association constants of the binuclear ligands [Pt(terpy)S[CH2]4S(terpy)Pt]2+ and [Pt(terpy)S[CH2]6S(terpy)Pt]2+ are 8 and 23 times larger respectively than the affinity of the monomer. For the latter binuclear derivative the increase may be ascribed to bifunctional reaction. Differential dialysis experiments with DNAs of differing base composition show that [Pt(terpy)SC4H9]+ has a requirement for a single G X C base-pair at the highest-affinity site. However, in the binuclear ligands chromophore specificity is severely compromised. Similar experiments indicate that 9-aminoacridine and selected methylene-linked diacridines show no significant sequence selectivity.