Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

A real time quantitative PCR-based method for the detection and quantification of simian virus 40.

A real time quantitative PCR-based simian virus 40 (SV40) detection and quantification method has been developed. This method takes advantage of the 5' to 3'-exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for direct monitoring of PCR product accumulation through a dual-labelled fluorogenic probe. This method provides accurate, precise and reproducible quantification of SV40 DNA over a linear dynamic range of at least 100,000-fold with a minimum detection level of 6.4 copy equivalents/microL of SV40 viral particle in test samples. The sample preparation procedure employed allows for efficient and consistent recovery of SV40 DNA from test samples. High concentrations of protein and cellular DNA presenting in test samples have been demonstrated to have no impact on SV40 quantification. This method offers significant advantages over other PCR methods and cell-based infectivity assays currently available for SV40 detection and quantification. The availability of this method should greatly facilitate the pathogenic investigation of SV40, as well as viral clearance evaluations required for the development of new biological products.     

A study of the dicyclohexylcarbodiimide-binding component of the mitochondrial ATPase complex from beef heart

1. The binding of [14C]-dicyclohexylcarbodiimide to membrane proteins of beef heart mitochondria has been investigated using dodecylsulphate/polyacrylamide gel electrophoresis. Upon incubation of submitochondrial particles with low concentrations of dicyclohexylcarbodiimide (5 nmol/mg protein) radioactivity was incorporated into three components with apparent molecular weights of 30000, 18000 and less than 6500. Only the two smaller components were found to be extracted into chloroform/methanol. The same two components were labelled when the isolated ATPase complex or a reconstituted F0F1 system was incubated with low concentrations of dicyclohexylcarbodiimide. High concentrations of dicyclohexylcarbodiimide (20-100 nmol/mg protein) resulted in binding to several mitochondrial proteins. 2. The maximal amount of dicyclohexylcarbodiimide which can bind to submitochondrial particles, the isolated ATPase complex, and the reconstituted F0F1 system was found to exceed the amount required for maximal inhibition of the ATPase activity by several-fold. The distribution of the bound [14C]dicyclohexylcarbodiimide between the different dicyclohexylcarbodiimide-binding components was investigated as a function of dicyclohexylcarbodiimide concentration. The smallest and largest components revealed a high affinity for dicyclohexylcarbodiimide-binding which paralleled the inhibition of ATPase activity. The intermediate component had a markedly lower affinity for dicyclohexylcarbodiimide-binding. 3. The larger dicyclohexylcarbodiimide-binding component of the isolated ATPase complex can be converted into the smaller component by treatment of the ATPase complex with performic acid. Partial conversion can also be achieved by extraction of the band from the dodecylsulphate-polyacrylamide gel after electrophoresis, followed by re-electrophoresis. The observations suggest that the larger component may be an oligomer of the smaller one. 4. Using concentrations of oligomycin and dicyclohexylcarbodiimide which were equal to or greater than those required for maximal inhibition of the ATPase activity, oligomycin was found to diminish the binding of [14C]dicyclohexylcarbodiimide to both dicyclohexylcarbodiimide-binding components of the isolated ATPase complex.     

Amount and turnover rate of the F0F1-ATPase and the stoichiometry of its inhibition by oligomycin in Rhodospirillum rubrum chromatophores

The amount of F1-ATPase in chromatophores from Rhodospirillum rubrum was determined by Western blotting using anti-RrF1 rabbit antibodies. 9.1 mmol F1 (mol bacteriochlorophyll)-1 was obtained or 14% of the total protein content of the chromatophores. The turnover rate of the F0F1-ATPase was 17 molecules ATP s-1 during synthesis, 2 molecules ATP s-1 during hydrolysis under coupled conditions with Mg2+ as the divalent cation, and 7 molecules ATP s-1 during hydrolysis in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Binding of 1 mol oligomycin/mol F0F1-ATPase was found to inhibit the activities of the enzyme completely. A single binding site was found with a Kd of approximately 2 microM.     

The Slr0924 protein of Synechocystis sp. strain PCC 6803 resembles a subunit of the chloroplast protein import complex and is mainly localized in the thylakoid lumen

An isolated 25 kDa protein of Synechocystis sp. PCC 6803 was N-terminally sequenced and assigned to a protein encoded by the ORF slr0924. This ORF shows a certain degree of sequence similarity to a subunit from the protein Translocon at the Inner envelope of pea Chloroplasts (Tic22). The deduced amino acid sequence of Slr0924 has a N-terminal extension, that contains two possible translational start points and two possible cleavage sites for leader peptidases. Immunostaining with an antibody raised to the over-produced protein revealed two cross-reacting forms, which probably correspond to a larger intermediate and the mature protein. Immunogold labelling of thin sections showed that the protein is located mainly in the thylakoid region. This result was verified by thylakoid membrane fractionation indicating that Slr0924 is a lumenal protein. The slr0924 gene product is essential for the viability of Synechocystis sp. PCC 6803 as shown by interposon mutagenesis. The merodiploid strain showed reduced photosynthetic activity compared to the wild-type. Furthermore, growth of the merodiploid strain was found to be completely inhibited after cultivation with glucose. Accordingly, the amount of the slr0924 gene product was regulated by glucose and light intensities in wild-type cells. The potential function of the protein in Synechocystis sp. PCC 6803 will be discussed.