An apolipoprotein AI mimetic peptide: membrane interactions and the role of cholesterol

The 18-amino acid amphipathic helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH(2) promotes the separation of cholesterol from the phospholipid, resulting in the formation of cholesterol crystallites, even at mole fractions of cholesterol as low as 0.3. The peptide exerts a greater degree of penetration into membranes of pure phosphatidylcholine in the absence of cholesterol than into bilayers of phosphatidylcholine and cholesterol. The circular dichroism spectrum of the peptide in buffer indicates that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remains helical over a larger concentration range. The peptide undergoes a thermal transition on heating. Cholesterol has little effect on the secondary structure of the peptide; however, increased Trp emission intensity in the absence of cholesterol indicates a deeper penetration of the helix upon removal of cholesterol from the membrane. The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of this peptide.     

Position-specific suppression and enhancement of HIV-1 integrase reactions by minor groove benzo[a]pyrene diol epoxide deoxyguanine adducts: implications for molecular interactions between integrase and substrates

The viral protein HIV-1 integrase is required for insertion of the viral genome into human chromosomes and for viral replication. Integration proceeds in two consecutive integrase-mediated reactions: 3'-processing and strand transfer. To investigate the DNA minor groove interactions of integrase relative to known sites of integrase action, we synthesized oligodeoxynucleotides containing single covalent adducts of known absolute configuration derived from trans-opening of benzo-[a]pyrene 7,8-diol 9,10-epoxide by the exocyclic 2-amino group of deoxyguanosine at specific positions in a duplex sequence corresponding to the terminus of the viral U5 DNA. Because the orientations of the hydrocarbon in the minor groove are known from NMR solution structures of duplex oligonucleotides containing these deoxyguanosine adducts, a detailed analysis of the relationship between the position of minor groove ligands and integrase interactions is possible. Adducts placed in the DNA minor groove two or three nucleotides from the 3'-processing site inhibited both 3'-processing and strand transfer. Inosine substitution showed that the guanine 2-amino group is required for efficient 3'-processing at one of these positions and for efficient strand transfer at the other. Mapping of the integration sites on both strands of the DNA substrates indicated that the adducts both inhibit strand transfer specifically at the minor groove bound sites and enhance integration at sites up to six nucleotides away from the adducts. These experiments demonstrate the importance of position-specific minor groove contacts for both the integrase-mediated 3'-processing and strand transfer reactions.     

Relationships between fish feeding guild and trophic structure in English lowland shallow lakes subject to anthropogenic influence: implications for lake restoration

The shallow lakes of Eastern England have been subject to intense anthropogenic pressures including nutrient enrichment and fish stocking. We sought to determine the relationships between fish community structure and other ecosystem characteristics in 28 of these lakes through classification of fish species into piscivorous, zooplanktivorous and benthivorous feeding guilds according to the literature. Canonical correspondence analysis produced clear associations between fish and ecosystem characteristics that generally agreed with other theoretical (e.g. the alternative stable states hypothesis) and empirical studies, but with some important differences. There was a striking lack of relationships between nutrients and other variables, indicating the importance of top-down rather than bottom-up processes as a structuring force in the generally eutrophic study lakes. The presence of submerged (and shoreline) vegetation was associated with a diverse assemblage of apparently co-existing piscivorous (principally pike Esox lucius) and zooplanktivorous species. Perch Perca fluviatilis, a significant predator in other studies, was unimportant and argued to be limited by water quality in the extremely shallow lakes. In contrast, the benthivorous fish guild (principally carp Cyprinus carpio, bream Abramis brama and tench Tinca tinca) essentially represented the inverse of the potential pelagic associations between piscivores/zooplanktivores and vegetation. The introduction of large benthivores to many study lakes could have precipitated a loss of submerged vegetation through direct uprooting during foraging, with the effect of simplifying the fish community being most acute where littoral vegetation was limited by other anthropogenic factors. It is implied that attempts to promote or restore submerged vegetation in these lakes would best target benthivorous species.     

Genomic signatures to guide the use of chemotherapeutics

Using in vitro drug sensitivity data coupled with Affymetrix microarray data, we developed gene expression signatures that predict sensitivity to individual chemotherapeutic drugs. Each signature was validated with response data from an independent set of cell line studies. We further show that many of these signatures can accurately predict clinical response in individuals treated with these drugs. Notably, signatures developed to predict response to individual agents, when combined, could also predict response to multidrug regimens. Finally, we integrated the chemotherapy response signatures with signatures of oncogenic pathway deregulation to identify new therapeutic strategies that make use of all available drugs. The development of gene expression profiles that can predict response to commonly used cytotoxic agents provides opportunities to better use these drugs, including using them in combination with existing targeted therapies.     

Ciprofloxacin-Induced Antibacterial Activity is Reversed by Vitamin E and Vitamin C

In the present study, we investigated the possible involvement of oxidative stress in ciprofloxacin-induced cytotoxicity against several reference bacteria including Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). Oxidative stress was assessed by measurement of hydrogen peroxide generation using a FACScan flow cytometer. The antibacterial activity of ciprofloxacin was assessed using the disk diffusion method and by measuring the minimum inhibitory concentration (MIC). Ciprofloxacin induced a dose-dependent antibacterial activity against all bacteria where the highest tested concentration was 100 ug/ml. Results revealed that E. coli cells were highly sensitive to ciprofloxacin (MIC = 0.21 μg/mL ± 0.087), P. aeruginosa and S. aureus cells were intermediately sensitive (MIC = 5.40 μg/mL ± 0.14; MIC = 3.42 μg/mL ± 0.377, respectively), and MRSA cells were highly resistant (MIC = 16.76 μg/mL ± 2.1). Pretreatment of E. coli cells with either vitamin E or vitamin C has significantly protected cells against ciprofloxacin-induced cytotoxicity. These results indicate the possible antagonistic properties for vitamins C or E when they are used concurrently with ciprofloxacin.     

Studies of Mg2+/Ca2+ complexes of naturally occurring dinucleotides: potentiometric titrations, NMR, and molecular dynamics

Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.     

Measuring sunscreen protection against solar-simulated radiation-induced structural radical damage to skin using ESR/spin trapping: development of an ex vivo test method

The in vitro star system used for sunscreen UVA-testing is not an absolute measure of skin protection being a ratio of the total integrated UVA/UVB absorption. The in vivo persistent-pigment-darkening method requires human volunteers. We investigated the use of the ESR-detectable DMPO protein radical-adduct in solar-simulator-irradiated skin substitutes for sunscreen testing. Sunscreens SPF rated 20+ with UVA protection, reduced this adduct by 40-65% when applied at 2 mg/cm(2). SPF 15 Organic UVA-UVB (BMDBM-OMC) and TiO(2)-UVB filters and a novel UVA-TiO(2) filter reduced it by 21, 31 and 70% respectively. Conventional broad-spectrum sunscreens do not fully protect against protein radical-damage in skin due to possible visible-light contributions to damage or UVA-filter degradation. Anisotropic spectra of DMPO-trapped oxygen-centred radicals, proposed intermediates of lipid-oxidation, were detected in irradiated sunscreen and DMPO. Sunscreen protection might be improved by the consideration of visible-light protection and the design of filters to minimise radical leakage and lipid-oxidation.     

Variable Responses of Lowland Tropical Forest Nutrient Status to Fertilization and Litter Manipulation

Predicting future impacts of anthropogenic change on tropical forests requires a clear understanding of nutrient constraints on productivity. We compared experimental fertilization and litter manipulation treatments in an old-growth lowland tropical forest to distinguish between the effects of inorganic nutrient amendments and changes in nutrient cycling via litterfall. We measured the changes in soil and litter nutrient pools, litterfall, and fine root biomass in plots fertilized with nitrogen (N), phosphorus (P), or potassium (K), and in litter addition and litter removal treatments during 7 years. Soil inorganic N and litter N increased in double-litter plots but not in N-fertilized plots. Conversely, litter P and soil pools of P and K increased in fertilized plots but not in the double-litter plots. Soil and litter pools of N and K decreased in the no-litter plots. Changes in litterfall with added nutrients or litter were only marginally significant, but fine root biomass decreased with both the litter and the K addition. Differences between the two experiments are mostly attributable to the coupled cycling of carbon and nutrients in litter. Increased nutrient inputs in litter may improve plant uptake of some nutrients compared to fertilization with similar amounts. The litter layer also appears to play a key role in nutrient retention. We discuss our findings in the context of possible impacts of anthropogenic change on tropical forests.     

Effect of the active site D25N mutation on the structure, stability, and ligand binding of the mature HIV-1 protease

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR(D25N)) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 +/- 0.09 microm) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR(D25N) in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 degrees C, respectively. Only minimal differences are observed in high resolution crystal structures of PR(D25N) complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH(2) (RPB), as compared with PR.DRV and PR.RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T(m) is smaller for PR(D25N) (6.2 degrees C) than for PR (8.7 degrees C). The T(m) of PR(D25N).DRV increases by only 3 degrees C relative to free PR(D25N), as compared with a 22 degrees C increase for PR.DRV, and the mutation increases the ligand dissociation constant of PR(D25N).DRV by a factor of approximately 10(6) relative to PR.DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.     

Freshwater acidification: effects on the early life stages of fish

Summary Over the last 20–30 years there has been a very extensive international research effort into freshwater acidification and the decline or loss of soft-water fisheries. The research includes many laboratory studies on physiology and survival of fish under the conditions caused by acidification. The causes of acidification have become increasingly well understood and accepted. In many countries, industrial and environmental management strategies are being implemented with the eventual aim of halting or reversing acidification in vulnerable areas. Attention has shifted away from research into the effects of acidification on fish, and the number of new papers on this subject is declining. Now appears a good time to review this research.As is often the case in research of this kind, it is difficult to compare work from different laboratories. Especially in earlier work, experimental conditions may not be fully relevant to the field (e.g. in the choice of ambient calcium concentration), experimental media may be inadequately specified (e.g. trace metal concentrations) and analytical techniques may be open to question (e.g. pH measurement). This may explain, in part, why agreement is lacking on many aspects of the physiological effects on the early life stages of fish, as well as the adult stages to some extent. If research is to continue in this field, then adoption of standard soft, acid water media would be of significant benefit. Nethertheless, some very general conclusions on fish early life stages can be reached.There is some evidence of impaired gametogenesis and spawning, but in this area there is still a need for systematic research. After fertilization, the chorionic membranes afford protection from the effects of low pH and especially the accompanying toxic trace metals, notably aluminium. Indeed, dissolved aluminium can ameliorate acid toxicity at this stage in development, possibly by helping to decrease membrane permeability and reduce H⁺ uptake. The main hazard seems to be impaired development (perhaps related to a decline in pH of the perivitelline fluid), often leading to a lack of ability to escape from the chorionic membranes on hatching.After hatching, vulnerability increases. Dissolved aluminium, and other trace metals associated with acidification, become more important. Some studies indicate continuing protection from low pH afforded by low concentrations of aluminium. The causes of death can still only be inferred: much attention has concentrated on mineral uptake and balance, and there is anecdotal evidence of respiratory impairment, although not as unequivocal as it is for postlarval fish. Comparison with the physiological effects on postlarval fish is not necessarily helpful. There seems to be at least one important difference. Unlike postlarvel fish, larvae do not find aluminium at around pH 5.5 (when respiratory effects are probably most important) the most hazardous combination; rather, the greatest danger to larvae seems to be when very low pH (<5.0) and trace metals (including aluminium) combine in some kind of joint toxic effect.There is a great increase in vulnerability once dependence on the yolk has come to an end. This period coincides with the movement from the incubation site in the substratum into the open water, where the relevant water chemistry is likely to fluctuate between greater extremes. However, it is earlier, during autumn rainstorms and spring snowmelt, that pH and concentrations of protective calcium ions usually reach their lowest and metals like aluminium their highest values. In waters where acidification is most likely to occur, the early life stages of many freshwater fish species will be close to their survival threshold and only a slight decline in water quality might result in the loss of a complete year class, which would affect recruitment and eventual population status.With gradual acceptance of the causes and effects of freshwater acidification, the research effort in relation to fish is declining or shifting towards attempts to re-establish fisheries in vulnerable areas. As outlined above, only very general conclusions can be reached on individual life stages, and the relative susceptibilities compared. However, in many respects the sequence of events in the loss of fisheries is not fully understood, and information is lacking on how events at significant stages of the life cycle affect subsequent survival, development and recruitment. Plenty of work is still required.