Dynamics of an oscillating viscoelastic sphere: a model of the vitreous humor of the eye

The vitreous humor of the eye is modelled as a homogeneous viscoelastic sphere. The analysis provides a relation for the displacement amplitude and phase following oscillatory and step excitations, as functions of the storage and loss moduli of the vitreous gel, and allows calculation of the resolution requirements for a non-invasive optically-based diagnostic method for the viscoelastic properties of the vitreous.     

Induction of mutations in the zebrafish with ultraviolet light.

Recessive lethal germline and specific locus somatic mutations were induced efficiently in the zebrafish by exposure of mature sperm to UV light. Mutagenesis of sperm yielded mosaic individuals: clones bearing novel mutations represented approximately 12-25% of the haploid germ cells and 25-50% of the somatic tissue. Simple methods are described for the reliable identification and propagation of newly arising developmental mutations in zebrafish.     

Informatic search strategies to discover analogues and variants of natural product archetypes

Natural products are a crucial source of antimicrobial agents, but reliance on low-resolution bioactivity-guided approaches has led to diminishing interest in discovery programmes. Here, we demonstrate that two in-house automated informatic platforms can be used to target classes of biologically active natural products, specifically, peptaibols. We demonstrate that mass spectrometry-based informatic approaches can be used to detect natural products with high sensitivity, identifying desired agents present in complex microbial extracts. Using our specialised software packages, we could elaborate specific branches of chemical space, uncovering new variants of trichopolyn and demonstrating a way forward in mining natural products as a valuable source of potential pharmaceutical agents.     

Osmoregulation in the Halotolerant Alga Asteromonas gracilis

Asteromonas gracilis, a green wall-less halotolerant alga, grows on salt concentrations from 0.5 molar NaCl (seawater) to saturation (4.5 molar NaCl). The specific growth rate was maximal at concentrations between 0.5 and 2.5 molar and only gradually decreased above 2.5 molar. Photosynthetic oxygen evolution was maximal over a range of salinities around 2.5 molar and the photosynthesis to respiration ratio showed a maximum at 1.5 molar NaCl. The alga accumulates large amounts of intracellular glycerol in response to saline conditions. The glycerol content of the cells varied in direct proportion to the extracellular salt concentration, being about 50 and 400 picograms glycerol per cell in algae grown at 0.5 and 4.5 molar NaCl, respectively. In salt concentrations lower than 3.5 molar and at growth temperatures below 40 C, essentially all the glycerol was intracellular. Above 3.5 molar NaCl, about 25 per cent of the total glycerol leaked slowly from the cells to the medium. Treating the algae for several minutes at temperatures exceeding 47 C caused 50 per cent release of the internal glycerol. At 60 C, 100 per cent of the glycerol was released. When the extracellular salt concentration was increased or decreased, the intracellular glycerol varied accordingly, reaching its new intracellular level after a few hours. Both photosynthesis and respiration were inhibited on transfer of the cells from 1.5 to 3.5 molar NaCl but were not inhibited on transfer of the cells from 3.5 to 1.5 molar NaCl. The kinetics of photosynthetic resumption preceded the kinetics of glycerol biosynthesis. The above results indicate the existence of osmotic regulations in Asteromonas gracilis via the accumulation of intracellular glycerol.     

Foliar Fatty Acids and Sterols of Soybean Field Fumigated with SO(2)

Sixty-day-old soybean plants were exposed in the field to 78.7 parts per one-hundred million of SO₂ in an open-air fumigation system for 20 days. Leaves from the top one-fourth and bottom one-fourth of the plants were analyzed for chlorophyll, free fatty acids, fatty acid esters, polar lipid fatty acids, and sterols. Fumigated plants had a lower chlorophyll, free fatty acid, and polar lipid content, but a higher fatty acid ester content. Of the individual fatty acids, linoleic and linolenic acid increased with SO₂ fumigation while palmitic acid decreased. SO₂ fumigations had only a minor effect on leaf sterols. In general, the lower, more mature leaves showed a greater response to SO₂ exposure.     

Thermodynamic limits to the ATP/site stoichiometries of oxidative phosphorylation by rat liver mitochondria

From measurements of reactants, products, and the oxidation-reduction state of cytochrome c + c1 during 3-hydroxybutyrate-supported oxidative phosphorylation by rat liver mitochondria at static head (state 4), we determined the free energy change of ATP formation from ADP and Pi (phosphorylation potential or delta GP) and the oxidation-reduction free energy changes (redox potentials or delta GR values) across Sites 1 + 2 (delta GR1 + 2), across Site 3 (delta GR3), and across Sites 1 + 2 + 3 (delta GR). At pH 7.4, -delta GR1 + 2/delta GP, -delta GR3/delta GP, and -delta GR/delta GP were maximally 1.80, 1.56, and 3.37. These can be taken as thermodynamic upper limits to the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometry of 3-hydroxybutyrate-supported oxidative phosphorylation. The theory of linear nonequilibrium thermodynamics were employed to estimate lower limits to the ATP/site stoichiometries. The lower limit is given by the expression, q2(-delta GRsite/delta GP). The degree of coupling, q, was 0.977 as determined from the dependence of respiratory rate on delta GP. Determined in this way, lower limits of the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometries were 1.67, 1.44, and 3.11, respectively. ADP addition to mitochondria incubated at static head lowered delta GP by 1.1 kcal/mol and stimulated respiration by a factor of about 2.5 but caused negligible changes in delta GR1 + 2 and delta GR3. This observation demonstrates that the respiratory reactions from substrate to cytochrome c and from cytochrome c to oxygen both move away from thermodynamic equilibrium with delta GP during the transition from resting to active oxidative phosphorylation. The findings are discussed in terms of current schemes of chemiosmotic coupling.     

Changes in 24-h substrate oxidation in older and younger men in response to exercise.

The purpose of this study was to compare 24-h substrate oxidation in older (OM; 60-75 yr, n = 7) and younger (YM; 20-30 yr, n = 7) men studied on sedentary day (Con) and on a day with exercise (Ex; net energy expenditure = 300 kcal). Plasma glucose and free fatty acids were also measured at several time points during the 24-h measurement. Weight was not different in OM and YM (means +/- SD; 84.8 +/- 16.9 vs. 81.4 +/- 10.4 kg, respectively), although percent body fat was slightly higher in OM (25.9 +/- 3.5 vs. 21.9 +/- 9.7%; P = 0.17).Values of 24-h energy expenditure did not differ in OM and YM on the Con (means +/- SE; 2,449 +/- 162 vs. 2,484 +/- 104 kcal/day, respectively) or Ex (2,902 +/- 154 vs. 2,978 +/- 122 kcal/day) days. Under both conditions, 24-h respiratory quotient was significantly lower and fat oxidation significantly higher in OM. Glucose concentrations were not different at any time point, but plasma free fatty acid concentrations were higher in OM, particularly following meals. Thus, under these controlled conditions, 24-h fat oxidation was not reduced and was in fact greater in OM. We speculate that differences in the availability of circulating free fatty acids in the postprandial state contributed to the observed differences in 24-h fat oxidation in OM and YM.     

Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications

Washed and purified rat- or mouse-liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine O and a cyanine derivative, 3,3'-dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non-energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non-energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non-specific dye binding) and a second population which exhibited high fluorescence. Flow cytometry of the unpurified, simply washed, rat-liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light-scattering properties and membrane-potential-related fluorescence. One third of the washed mitochondria were found to be non-functional by such assays. The fluorescence of purified rat-liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogenous substrates. The low-angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization. The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non-active mitochondrial population dispersion) point of view could thus be demonstrated by flow-cytometry analysis. Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat-liver mitochondria), and the effects of ammonium toxicity (mouse-liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies.     

Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase

Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane α-helices coordinating two hemes. We studied the role of the second transmembrane α-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.