Malignancy-associated DNA-binding protein C3DP from human serum. Further characterization and purification of C3DP retaining its DNA-binding affinity.

C3DP, a malignancy-associated DNA-binding protein from human serum[1], was purified to homogeneity without loss of its DNA-binding affinity. For this purpose normal human serum was submitted to affinity chromatography on Con A-Sepharose and DNA-cellulose and to preparative polyacrylamide gel electrophoresis. The purified C3DP was identified by immunodiffusion and sodium dodecylsulfate polyacrylamide gel electrophoresis and it was shown to bind to DNA by DNA-cellulose chromatography. The isoelectric point of C3DP was determined to 4.9 by isoelectric focusing.     

Immunological and reconstitution studies on the adenosine triphosphatase complex from Rhodospirillum rubrum

Studies on restoration of membrane-bound adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from Rhodospirillum rubrum show that the δ-subunit is capable of binding to the F₁ factor or to the F₀ moiety of the F₀-F₁ ATPase complex. This subunit is thus likely involved in linking the F₀ and F₁ factor.During solubilization of the oligomycin-sensitive F₀-F₁ ATPase complex with Triton X-100 the detergent becomes specifically associated with the lipophilic F₀ part of the enzyme complex.Crossed immunoelectrophoresis, agglutination tests, and kinetic studies with anti-F₁ ATPase antibodies reveal a reaction of immunological identity of membrane-bound ATPase, F₀-F₁ ATPase, and F₁ ATPase.     

Immunological properties of membrane-bound adenosine triphosphatase: immunological identification of rutamycin-sensitive F0.F1ATPase from Micrococcus luteus ATCC 4698 established by crossed immunoelectrophoresis.

(1) F0.F1ATPase (EC 3.6.1.3) from Micrococcus luteus ATCC 4698 was solubilized from plasma membranes by the non-ionic detergent Triton X-100 in the presence of 0.05 M MgCl2. (2) The antibiotics rutamycin, Dio-9, quercetin, oligomycin, botrycidin, efrapeptin, leucinostatin, valinomycin, and venturicidin as well as N,N'-dicyclohexylcarbodiimide and dinitrophenol are potent inhibitors of F0.F1ATPase activity.(3) F0.F1ATPase activity is completely inhibited by anti-F1ATPase antibodies. The inhibition is non-competitive. (4) Crossed immunoelectrophoresis reveals a reaction of immunological identity of F0.F1ATPase and F1ATPase indicating that both enzymes have in common antigenic sites.     

Kinetic studies on bacterial plasma membrane ATPase (F1). Nucleotide-induced long term inactivation of ATP hydrolyzing activity is linked to the formation of multiple "tight" enzyme nucleotide complexes.

ADP and the ATP analogs Nb-S6ITP (6-[(3-carboxy-4-nitrophenyl)thio]-9-beta-D-ribofuranosylpurine 5'-triphosphate) and AMP-P(NH)P (adenyl-5'-yl imidodiphosphate) interact with soluble plasma membrane ATPase (F1) from Micrococcus species in two ways: (i) at short incubation times, these inhibitors exhibit the kinetics of competitive inhibition, (ii) at long incubation times, these inhibitors induce an inactivation of the ATPase which can be reversed only in the case of AMP-P(NH)P. Kinetic treatment of the long term inactivation by ADP or Nb-S6ITP reveals a pseudo-first order process via the formation of an enzyme-inhibitor complex for which a Km analogous constant is obtained that is identical with the corresponding Ki value of the competitive inhibition. The long term inactivation by ADP and Nb-S6ITP involves the successive "tight" binding of 6 +/- 1 nucleotides/F1 molecule. One additional ADP molecule/F1 complex which is also "tightly" bound has no effect on the ATPase activity. The long term inactivation by ADP and Nb-S6ITP is inhibited at higher inhibitor concentrations according to a kinetics analogous to a substrate excess inhibition. Evidence is presented indicating that the mechanism of ATP hydrolysis by F1 and the long term inactivation by ADP or Nb-S6ITP are related processes. The mechanism of long term inactivation by AMP-P(NH)P appears to be different from that of ADP or Nb-S6ITP.     

Glycogen is the primary source of glucose during the lag phase of E. coli proliferation

In the studies of Escherichia coli (E. coli), metabolomics analyses have mainly been performed using steady state culture. However, to analyze the dynamic changes in cellular metabolism, we performed a profiling of concentration of metabolites by using batch culture. As a first step, we focused on glucose uptake and the behavior of the first metabolite, G6P (glucose-6-phosphate). A computational formula was derived to express the glucose uptake rate by a single cell from two kinds of experimental data, extracellular glucose concentration and cell growth, being simulated by Cell Illustrator. In addition, average concentration of G6P has been measured by CE-MS. The existence of another carbon source was suggested from the computational result. After careful comparison between cell growth, G6P concentration, and the computationally obtained curve of glucose uptake rate, we predicted the consumption of glycogen in lag phase and its accumulation as an energy source in an E. coli cell for the next proliferation. We confirmed our prediction experimentally. This behavior indicates the importance of glycogen participation in the lag phase for the growth of E. coli. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.     

Electrical and electronic components in the automotive sector: Economic and environmental assessment

Background Aims and Scope Automotive electrical and electronic systems (EES) comprise an area that has grown steadily in importance in the past decade and will continue to gain relevance in the foreseeable future. For this reason, the SEES project (Sustainable Electrical & Electronic System for the Automotive Sector) aims to contribute to cost-effective and eco-efficient EES components. Scenarios for the recovery of automotive EES are defined by taking into consideration the required improvements in EES design and the development and implementation of new technologies. The research project SEES is funded by the European Commission (Contract no. TST3-CT-2003-506075) within the Sixth Framework Programme, priority 6.2 (see 〈www.sees-project.net〉 for more information). This paper presents the findings of an assessment of the environmental and economic improvements for automotive EES from a system perspective, taking into account all life cycle steps. Methods Life Cycle Assessment (LCA) and Life Cycle Costing (LCC) case studies have been employed within the SEES project to define optimum design and end-of-life scenarios. These case studies have been applied to two selected EES components: an engine wire harness and a smart junction box, both manufactured by LEAR and assembled in an existing Ford car model. The component design has a significant impact on the product system and its processes, including its use and end-of-life (EOL) phase. For each of the analysed components, two potential design alternatives have been compared with the original design, based on designers’ recommendations from the status quo scenario results. These include the use of alternative wiring systems with a reduced copper content (flat flexible cable), lead-free solder alloys and new fixation mechanisms to facilitate disassembly. The overall EOL scenario determines the technologies of processes that must be modelled within the EOL phase of a product system. The analysed end-of-life scenarios include: status quo car recycling and two alternatives: 1. disassembly for specific EES component recycling; 2. advanced post-shredder recycling of shredding residues. The influences of the different design and EOL treatment scenarios on the LCA and LCC results have been analysed. Results The most dominant life cycle phases for the LCA results are manufacturing (including raw material extraction and manufacturing of materials and components) and the use-phase. Similarly, manufacturing was the predominant phase during the LCC study. Disassembly costs were shown to be significant during the EOL phase. Among the analysed design alternatives, the highest environmental improvement potential were gained from the use of alternative wiring systems with reduced weight and copper content, but with slightly increased life cycle costs. Smaller differences of the results were determined for the different end-of-life scenarios. Discussion The results of the EOL scenario depend on the component in question. The influence of variations in process data, model choices, e.g. which LCIA model was used for calculating the Human Toxicity Potential (HTP), which inventory data for copper production was used and other variables have been assessed in the sensitivity analysis. The sensitivity analysis demonstrates a strong dependency of results for HTP on the selected model. The presented results are based on a public report of the SEES project. The study has undergone a critical review by an external expert according to ISO 14040, § 7.3.2. Conclusions The environmental impacts during the life cycle of the analysed products are generally most strongly influenced by material production and the use phase of the products. In comparison, improvements during the EOL phase have only a very limited potential to reduce environmental impacts. The studied design changes displayed clear environmental advantages for (lighter) flat, flexible cables. Whereas, the lead-free solder design alternatives showed a slight increase in some environmental impact categories. The application of these design changes has been limited in some cases by technical issues. Recommendations and Perspectives Focussing only on end-of-life improvements cannot be recommended for automotive EES products. A life-cycle perspective should be utilised for assessing improvements in individual life cycle stages of a product. The presented results will be an input for Eco-design guidelines for automotive EES, to be developed at a later stage within the SEES project. ESS-Submission Editor: Dr. Lester Lave (II01@andrew.cmu.edu)     

Ernst Haeckel's concept of an evolutionary origin of life

In 1865/66 E. Haeckel for the first time suggested an evolutionary sequence in order to explain the origin of the first living cell. Haeckel's concept is compared with modern theories of the origin of life. It is evident that Haeckel has not as yet received the credit that he deserves for his evolutionary concept.