A Three-Dimensional Skeletal Reconstruction of the Stem Amniote Orobates pabsti (Diadectidae): Analyses of Body Mass,Centre of Mass Position,and Joint Mobility

Orobates pabsti, a basal diadectid from the lower Permian, is a key fossil for the understanding of early amniote evolution. Quantitative analysis of anatomical information suffers from fragmentation of fossil bones, plastic deformation due to diagenetic processes and fragile preservation within surrounding rock matrix, preventing further biomechanical investigation. Here we describe the steps taken to digitally reconstruct MNG 10181, the holotype specimen of Orobates pabsti, and subsequently use the digital reconstruction to assess body mass, position of the centre of mass in individual segments as well as the whole animal, and study joint mobility in the shoulder and hip joints. The shape of most fossil bone fragments could be recovered from micro-focus computed tomography scans. This also revealed structures that were hitherto hidden within the rock matrix. However, parts of the axial skeleton had to be modelled using relevant isolated bones from the same locality as templates. Based on the digital fossil, mass of MNG 10181 was estimated using a model of body shape that was varied within a plausible range to account for uncertainties of the dimension. In the mean estimate model the specimen had an estimated mass of circa 4 kg. Varying of the mass distribution amongst body segments further revealed that Orobates carried most of its weight on the hind limbs. Mostly unrestricted joint morphology further suggested that MNG 10181 was able to effectively generate propulsion with the pelvic limbs. The digital reconstruction is made available for future biomechanical studies.     

Mitotic chromosome length scales in response to both cell and nuclear size

Multicellular development requires that cells reduce in size as a result of consecutive cell divisions without increase in embryo volume. To maintain cellular integrity, organelle size adapts to cell size throughout development. During mitosis, the longest chromosome arm must be shorter than half of the mitotic spindle for proper chromosome segregation. Using high-resolution time-lapse microscopy of living Caenorhabditis elegans embryos, we have quantified the relation between cell size and chromosome length. In control embryos, chromosome length scaled to cell size. Artificial reduction of cell size resulted in a shortening of chromosome length, following a trend predicted by measurements from control embryos. Disturbing the RAN (Ras-related nuclear protein)-GTP gradient decoupled nuclear size from cell size and resulted in chromosome scaling to nuclear size rather than cell size; smaller nuclei contained shorter chromosomes independent of cell size. In sum, quantitative analysis relating cell, nuclear, and chromosome size predicts two levels of chromosome length regulation: one through cell size and a second in response to nuclear size.     

Sulfate transport by rat liver lysosomes

Sulfate transport was examined using membrane vesicles (pH 7.0 inside) prepared from rat liver lysosomes. Sulfate uptake was dependent upon external pH with increased uptake at lower buffer pH. The Km for uptake was 160 microM at pH 5.0 while at pH 7.0, a lower affinity system with a Km of 1.4 mM was present. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased uptake at pH 5.0 while valinomycin/KCl had no effect. In contrast, at pH 7.0, valinomycin-induced changes in membrane potential stimulated uptake. Countertransport of sulfate at pH 7.0 was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid, and a variety of anions: SO4(2-) greater than MoO4(2-) greater than Cl- greater than HPO4- greater than HCO3-. Trans-stimulation of sulfate uptake at pH 7.0 was observed with MoO4(2-) and, to a lesser extent, with S2O3(2-) while Cl-, HPO4-, and HCO3- had little effect. However, chloride loading of vesicles resulted in marked stimulation of sulfate uptake at pH 5.0. It appears that sulfate and protons exit lysosomes in exchange for chloride by a specific, pH-regulated anion transport system.     

Modification of the in vitro Cytotoxicity of Hydrogen Peroxide by Iron Complexes

The effect of a range of iron chelates on the cytotoxicity of H₂O₂ was studied on a mammalian epithelial cell line. Iron complexes which were internalised enhanced the cytotoxicity of H₂O₂ measured by delayed thymidine incorporation. Iron complexed to 8-hydroxyquinoline (Fe/8-HQ) potentiated the cytotoxicity of 50 µM by 38% and Fe/dextran by 23%. Pre-exposure of cells to Fe/dextran at 4°C did not result in any potentiation of H₂O₂-induced cytotoxicity which we ascribe to failure of the Fe/dextran to be endocytosed at low temperature. Iron complexes which are slowly taken up or remain extracellular protected the cells from H₂O₂-induced cytotoxicity. Thus, Fe/EDTA inhibited the cytotoxicity of 50 µM H₂O₂ by 33%; Fe/ADP by 80% and Fe/ATP by 88%, suggesting mutual extracellular detoxification.     

Kinetics of lecithin-cholesterol acyltransferase reaction with discoidal complexes of apolipoprotein A-I.phosphatidylcholine.ether phospholipid.cholesterol

Discoidal substrates for purified human lecithin-cholesterol acyltransferase were prepared with human apolipoprotein A-I, cholesterol, and egg phosphatidylcholine (PC) or dipalmitoyl PC, including dihexadecyl PC in various proportions as an enzymatically inert dilutor of the interfacial PC substrate. All the complexes, prepared by the sodium cholate dialysis method, were found to be very similar in size, lipid/apolipoprotein stoichiometry, and apolipoprotein spectral properties to the small discoidal complexes without any dihexadecyl PC, described previously (Jonas, A., and Matz, C.E. (1982) Biochemistry 21, 6867-6872; Jonas, A., and McHugh, H. T. (1984) Biochim. Biophys. Acta 794, 361-372). The kinetic results presented in the form of double reciprocal plots of initial velocity against bulk PC or interfacial PC concentration were linear according to the Verger et al. kinetic model (Verger, R., Mieras, M. C. E., and de Haas, G. H. (1973) J. Biol. Chem. 248, 4023-4034) for an initial enzyme binding via an interfacial recognition site followed by interfacial substrate binding and catalysis, in the presence of a competitive interfacial inhibitor. The results indicate, furthermore, that the affinity of the active site for the substrate and inhibitor is quite similar.     

Long-term development in a Baltic fish community exposed to bleached pulp mill effluent

Observations near a Swedish pulp mill at the Bothnian Sea during 1982-1992 revealed low fish abundance and a disturbed community structure. The presence of endocrine disrupting or toxic substances in effluent water was indicated by biomarker responses, impaired reproduction and stimulated growth in perch (Perca fluviatilis). In 1992 the bleaching process was altered and a secondary treatment system was installed, improving the quality and decreasing the amount of effluent. This paper presents the results of follow-up studies during 1995-1998 with the objective to analyse any recovery of the fish community due to changes in mill operations. Although abundance and diversity of the fish community did recover, an effect of enrichment, indicated by high catches of cyprinids, was still visible in the vicinity of the mill outlet. Sexual maturation was delayed, gonad development was retarded, average growth rate was faster, and the condition factor was higher in perch caught in the vicinity of the effluent outlet, than in perch captured elsewhere. It is concluded that substances inhibiting reproduction and causing growth disturbance were still present in the effluents in effective concentrations.