Comparison of proteins from Mycobacterium fortuitum, Mycobacterium nonchromogenicum and Mycobacterium terrae using flat bed electrophoresis.

Polyacrylamide gel electrophoresis of bacterial lysates in a flat bed gives a linear relationship between 1n mol. wt of the proteins and the square root of their migration distances, thereby allowing standardization of different electrophoresis runs and precise comparison between homologous bands. The results obtained with Mycobacterium fortuitum, M. terrae and M. nonchromogenicum strains were used in numerical analysis. Mycobacterium fortuitum and M. nonchromogenicum showed a greater internal similarity than M. terrae, while two strains of the latter clustered with M. nonchromogenicum. The method described allows the comparison of mycobacteria with different generation times and provides a large number of good characters for numerical taxonomy.     

Quantification of short term signaling by the epidermal growth factor receptor.

During the past decade, our knowledge of molecular mechanisms involved in growth factor signaling has proliferated almost explosively. However, the kinetics and control of information transfer through signaling networks remain poorly understood. This paper combines experimental kinetic analysis and computational modeling of the short term pattern of cellular responses to epidermal growth factor (EGF) in isolated hepatocytes. The experimental data show transient tyrosine phosphorylation of the EGF receptor (EGFR) and transient or sustained response patterns in multiple signaling proteins targeted by EGFR. Transient responses exhibit pronounced maxima, reached within 15-30 s of EGF stimulation and followed by a decline to relatively low (quasi-steady-state) levels. In contrast to earlier suggestions, we demonstrate that the experimentally observed transients can be accounted for without requiring receptor-mediated activation of specific tyrosine phosphatases, following EGF stimulation. The kinetic model predicts how the cellular response is controlled by the relative levels and activity states of signaling proteins and under what conditions activation patterns are transient or sustained. EGFR signaling patterns appear to be robust with respect to variations in many elemental rate constants within the range of experimentally measured values. On the other hand, we specify which changes in the kinetic scheme, rate constants, and total amounts of molecular factors involved are incompatible with the experimentally observed kinetics of signal transfer. Quantitation of signaling network responses to growth factors allows us to assess how cells process information controlling their growth and differentiation.     

Denitrification with methanol in the presence of high salt concentrations and at high pH levels

Summary In the combined ion exchange/biological denitrification process for nitrate removal from ground water, in which nitrate is removed by ion exchange, the resins are regenerated in a closed circuit by a biological denitrification reactor. This denitrification reactor eliminates nitrate from the regenerant. Methanol is used as electron donor for biological denitrification. To obtain sufficient regeneration of the resins within a reasonable time, high NaCl or NaHCO₃ concentrations (10–30 g/l) in the regenerant are necessary. High NaHCO₃ concentrations affected the biological denitrification in three ways: a) a slight decrease in denitrification capacity (30%) was observed; b) the yield coefficient and CH₃OH/NO₃ ^-–N ratio decreased. When high NaHCO₃ concentrations (above 10g NaHCO₃/l) were used, the yield coefficient was 0.10–0.13 g VSS/g NO₃ ^-–N and the CH₃OH/NO₃ ^-–N ratio was 2.00–2.03 g/g; c) high NaHCO₃ concentrations influenced nitrite production. Nitrite is an intermediate product of biological denitrification and with rising NaHCO₃ concentrations nitrite accumulation was suppressed. This was explained by the effect of high NaHCO₃ concentrations on the pH in the microenvironment of the denitrifying organisms. High NaCl concentrations also resulted in a slight decrease in denitrification capacity, but the second and third effects were not observed in the presence of high NaCl concentrations.Although the pH in the regenerant will rise as a result of biological denitrification, the capacity of a denitrification reactor did not decrease significantly when a pH of 8.8–9.2 was reached.     

Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and mixed bilayers

Coat protein of bacteriophage M13 is examined in micelles and vesicles by time-resolved tryptophan fluorescence and anisotropy decay measurements and circular dichroism experiments. Circular dichroism indicates that the coat protein has alpha-helix (60%) and beta-structure (28%) in 700 mM sodium dodecyl sulfate micelles and predominantly beta-structure (94%) in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidic acid (80/20 w/w) small unilamellar vesicles. The fluorescence decay at 344 nm of the single tryptophan in the coat protein after excitation at 295 or 300 nm is a triple exponential. In the micelles the anisotropy decay is a double exponential. A short, temperature-independent correlation time of 0.5 +/- 0.2 ns reflects a rapid depolarization process within the coat protein. The overall rotation of the coat protein-detergent complex is observed in the decay as a longer correlation time of 9.8 +/- 0.5 ns (at 20 degrees C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In vesicles at all lipid to protein molar ratios in the range from 20 to 410, the calculated order parameter is constant with a value of 0.7 +/- 0.1 from 10 to 40 degrees C, although the lipids undergo the gel to liquid-crystalline phase transition. The longer correlation time decreases gradually on increasing temperature. This effect probably arises from an increasing segmental mobility within the coat protein. The results are consistent with a model in which the coat protein has a beta-structure and the tryptophan indole rings do not experience the motion of the lipids in the bilayer because of protein-protein aggregation.     

Time-resolved fluorescence spectroscopy of lumazine protein from Photobacterium phosphoreum using synchrotron radiation

Time-resolved fluorescence on lumazine protein from Photobacterium phosphoreum was performed with synchrotron radiation as a source of continuously tunable excitation. The experiments yielded structural and dynamic details from which two aspects became apparent. From fluorescence anisotropy decay monitoring of lumazine fluorescence with different excitation wavelengths, the average correlation times were shown to change, which must indicate the presence of anisotropic motion of the protein. A similar study with 7-oxolumazine as the fluorescent ligand led to comparable results. The other remarkable observation dealt with the buildup of acceptor fluorescence, also observed with 7-oxolumazine although much less pronounced, which is caused by the finite energy transfer process between the single donor tryptophan and the energy accepting lumazine derivatives. Global analytical approaches in data analysis were used to yield realistic correlation times and reciprocal transfer rate constants. It was found that the tryptophan residue has a large motional freedom as also reported previously for this protein and for the related protein from P. leiognathi (Lee et al. 1985; Kulinski et al. 1987). The average distance between the tryptophan residue and the ligand donor-acceptor couple has been determined to be 2.7 nm for the same donor and two different acceptors.     

The emergence of a new chlorophytan system,and Dr. Kornmann's contribution thereto

In traditional chlorophytan systems the organizational level was the primary character for the distinction of main groups (classes and orders). For instance, in Fott (1971), the flagellate level corresponds with the Volvocales, the coccoid level with the Chlorococcales, the filamentous level with the Ulotrichales, the siphonocladous level with the Siphonocladales, and the siphonous level with the Bryopsidales. The new system presented here is an elaboration and emendation of recently proposed taxonomies and their underlying phylogenetic hypotheses, and it is mainly based on ultrastructural features which have become available over the last 15 years. The following criteria are used for the distinction of classes and orders: (1) architecture of the flagellate cell (flagellate cells are considered as the depositories of primitive characters); (2) type of mitosis-cytokinesis; (3) place of meiosis in the life history and, consequently, the sexual life history type; (4) organizational level and thallus architecture; (5) habitat type (marine versus feshwater and terrestrial); (6) chloroplast type. The following classes are presented: Prasinophyceae, Chlamydophyceae, Ulvophyceae (orders Codiolales, Ulvales, Cladophorales, Bryopsidales, Dasycladales), Pleurastrophyceae (?), Chlorophyceae s.s. (orders Cylindrocapsales, Oedogoniales, Chaetophorales), Zygnematophyceae, Trentepohliophyceae, Charophyceae (orders Klebsormidiales, Coleochaetales, Charales). The new system no longer reflects the traditional hypothesis of a stepwise evolutionary progression of organizational levels in which the flagellate level represents the most primitive lineage, the coccoid and sarcinoid levels lineages of intermediate derivation, and the filamentous, siphonocladous and siphonous levels the most derived lineages. Instead, it is now hypothesized that these levels have arisen over and over again in different chlorophytan lineages which are primarily characterized by their type of flagellate cell. The flagellate green algal classes Prasinophyceae (with organic body scales) and Chlamydophyceae probably represent bundles of highly conservative lineages that diverged very long ago. Consequently, extant genera and species in these classes can be expected to have emerged long ago. Fossil evidence points to a minimum age of 600 Ma of certain extant Prasinophycean genera, and molecular evidence to a minimum age of 400–500 Ma of a fewChlamydomonas species. On the contrary, the most derived “green algal” lineage, the Angiosperms, can be expected to consist of, on average, much younger genera and species. Fossil evidence points to a minimum age of genera of 5–60 Ma. Lineages of intermediate evolutionary derivation (Ulvophyceae, Chlorophyceae, Charophyceae) can be expected to encompass genera and species of intermediate age. Fossil and (limited) molecular evidence point to a minimum age of 230–70 Ma of extant genera in Bryopsidales, Dasycladales and Cladophorales (Ulvophyceae) and of 250–80 Ma of extant genera in Charales (Charophyceae).     

Lycorine: a eukaryotic termination inhibitor?

The effect of the alkaloid lycorine on viral protein synthesis was studied in poliovirus-infected HeLa cells. The incorporation of [3H]leucine was inhibited by lycorine in a dose-dependent way, although lycorine never completely abolished translation. Using polyacrylamide gel electrophoresis, the viral proteins were identified as derived from the P1 (5' terminal), P2 (middle), or P3 (3' terminal) region of the poliovirus translation unit. The residual labeling of viral proteins in the presence of lycorine was mainly due to synthesis of P1 proteins and slightly less to P2 proteins, while virtually no P3-derived proteins were made. It is suggested that lycorine may act at the level of termination.