Stabilities of hydroxyl radical spin adducts of PBN-type spin traps.

The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.     

Oxidation of cyanide to the cyanyl radical by peroxidase/H2O2 systems as determined by spin trapping

The cyanyl radical was formed during the oxidation of potassium or sodium cyanide by horseradish peroxidase, lactoperoxidase, chloroperoxidase, NADH peroxidase, or methemoglobin in the presence of hydrogen peroxide. The spin adducts of the cyanyl radical with 5,5-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-alpha-phenylnitrone were quite stable at neutral pH. The identity of these spin adducts could be demonstrated using 13C-labeled cyanide and by comparison with the spin adducts of the formamide radical, a hydrolysis product of the cyanyl radical adduct. The enzymatic conversion of cyanide to cyanyl radical by peroxidases should be considered in addition to its well-known role as a metal ligand. Furthermore, since cyanide is used routinely as an inhibitor of peroxidases, some consideration should be given to the biochemical consequences of this formation of the cyanyl radical by the catalytic activity of these enzymes.     

Secretion and glycosylation of alpha-foetoprotein by the mouse yolk sac.

Secretion and glycosylation of alpha-foetoprotein (AFP) by mouse yolk sac were studied by using yolk-sac explants cultured in vitro. Yolk-sac explants rapidly incorporated [35S]methionine into AFP, whereas radioactively labelled AFP was not found in the medium until 30 min after incubation was initiated. Electrophoretic analysis revealed that microheterogeneity of AFP synthesized in explants increased in parallel with the gestational age of the yolk sacs. The change in microheterogeneity was noted by the formation of increasingly acidic forms. Only the most acidic forms of AFP were found to be present in the medium on each gestational day studied. Tunicamycin reduced the incorporation of glucosamine into AFP with a concomitant decrease in molecular weight and microheterogeneity. However, the relative amount of AFP released into the medium was not altered by the presence of tunicamycin. The presence of under-glycosylated AFP in the medium indicates that glycosylation of AFP is not essential for its secretion from the yolk sac. In light of these and previous findings, it is suggested that the glycosylation of AFP may be important for the turnover of this glycoprotein in serum.     

Effects of the egg incubation environment on turtle carapace development

Developing organisms are often exposed to fluctuating environments that destabilize tissue-scale processes and induce abnormal phenotypes. This might be common in species that lay eggs in the external environment and with little parental care, such as many reptiles. In turtles, morphological development has provided striking examples of abnormal phenotypic patterns, though the influence of the environment remains unclear. To this end, we compared fluctuating asymmetry, as a proxy for developmental instability, in turtle hatchlings incubated in controlled laboratory and unstable natural conditions. Wild and laboratory hatchlings featured similar proportions of supernumerary scales (scutes) on the dorsal shell (carapace). Such abnormal scutes likely elevated shape asymmetry, which was highest in natural nests. Moreover, we tested the hypothesis that hot and dry environments cause abnormal scute formation by subjecting eggs to a range of hydric and thermal laboratory incubation regimes. Shape asymmetry was similar in hatchlings incubated at five constant temperatures (26–30°C). A hot (30°C) and severely Dry substrate yielded smaller hatchlings but scutes were not overtly affected. Our study suggests that changing nest environments contribute to fluctuating asymmetry in egg-laying reptiles, while clarifying the conditions at which turtle shell development remains buffered from the external environment.     

Visualizing and quantifying natural selection

Modern methods of analysis are enabling researchers to study natural selection at a new level of detail. Multivariate statistical techniques can Identify specific targets of selection and provide parameter estimates that fit into equations for evolutionary change. A more Intuitive understanding of the form of selection can be provided through graphical representation of selection surfaces. Combinations of quantitative and visual analyses are providing researchers with new insights into the details of natural selection in the wild.     

Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane.

Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical. The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl4. A mechanism is proposed for the formation of the trichloromethyl radical from CCl4 or BrCCl3.     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Effects of decreased atmospheric deposition on the sulfur budgets of two Dutch moorland pools

The chemical composition of surface waters of two Dutch moorland pools and of incident precipitation, was monitored from 1982 to 1990. For this period, sulfur and water budgets were calculated using a hydrochemical model developed for well-mixed non-stratifying lakes. Total atmospheric deposition of S decreased significantly after 1986 at both locations. A model describing the sulfur budget in terms of input, output and reduction/oxidation processes predicted a fast decrease of pool water SO₄ ²⁻ concentrations after a decrease of atmospheric input. However, SO₄ ²⁻ concentrations in the surface water was lowered only slightly or remained constant. Apparently a source within the lake caused the unexpectedly high SO₄ ²⁻ concentrations. The possible supply of SO₄ ²⁻ from the sediment through regulation by (K-)Al-SO₄ containing minerals or desorption of SO₄ ²⁻ from positively charged surfaces in the sediment was evaluated. Solubility calculations of pore water with respect to alunite, basaluminite and jurbanite indicated that SO₄ ²⁻ concentration was not regulated by these minerals. It is suggested here (1) that desorption of SO₄ ²⁻ from peaty sediments may account for the estimated SO₄ ²⁻ supply provided that the adsorption complex is periodically recharged by partial oxidation of the upper bottom sediments and (2) that because of exposure of a part of the pool bottom to the atmosphere during dry summers and subsequent oxidation of reduced S, the amount of SO₄ ²⁻ may be provided which complements the decreasing depositional SO₄ ²⁻ input. In future research these two mechanisms need to be investigated.