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).     

Temperature responses of tropical to warm temperateCladophora species in relation to their distribution in the North Atlantic Ocean

The relationship between distribution boundaries and temperature responses of some North AtlanticCladophora species (Chlorophyta) was experimentally examined under various regimes of temperature, light and daylength. Experimentally determined critical temperature intervals, in which survival, growth or reproduction was limited, were compared with annual temperature regimes (monthly means and extremes) at sites inside and outside distribution boundaries. The species tested belonged to two phytogeographic groups: (1) the tropical West Atlantic group (C. submarina: isolate from Curaçao) and (2) the amphiatlantic tropical to warm temperate group (C. prolifera: isolate from Corsica;C. coelothrix: isolates from Brittany and Curaçao; andC. laetevirens: isolates from deep and shallow water in Corsica and from Brittany). In accordance with distribution from tropical to warm temperate regions, each of the species grew well between 20–30°C and reproduction and growth were limited at and below 15°C. The upper survival limit in long days was <35°C in all species but high or maximum growth rates occurred at 30°C.C. prolifera, restricted to the tropical margins, had the most limited survival at 35°C. Experimental evidence suggests thatC. submarina is restricted to the Caribbean and excluded from the more northerly American mainland and Gulf of Mexico coasts by sporadic low winter temperatures in the nearshore waters, when cold northerly weather penetrates far south every few years. Experimental evidence suggests thatC. prolifera, C. coelothrix andC. laetevirens are restricted to their northern European boundaries by summer temperatures too low for sufficient growth and/or reproduction. Their progressively more northerly located boundaries were accounted for by differences in growth rates over the critical 10–15°C interval.C. prolifera andC. coelothrix are excluded or restricted in distribution on North Sea coasts by lethal winter temperatures, again differences in cold tolerance accounting for differences in their distribution patterns. On the American coast, species were probably restricted by lethal winter temperatures in the nearshore and, in some cases, by the absence of suitable hard substrates in the more equable offshore waters. Isolates from two points along the European coast (Brittany, Corsica) ofC. laetevirens showed no marked differences in their temperature tolerance but the Caribbean and European isolates ofC. coelothrix differed markedly in their tolerance to low temperatures, the lethal limit of the Caribbean isolate lying more than 5°C higher (at ca 5°C).     

NOTES ON CLADOPHORA (CHLOROPHYCEAE). II. CLADOPHORA CATENATA (LINNAEUS) KÜTZ., 1843, EMEND. V. D. HOEK, 1963

The species name Cladophora catenata (L.) Kütz. emend, van den Hoek has priority over Cladophora fuliginosa Kütz.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

Fluorescent probes located in heterogeneous environments give rise to anomalous time-resolved fluorescence anisotropy. A simple analytical expression of anisotropy has been derived for the case of a small difference in local fluorescence lifetimes. The expression has the diagnostic advantage that the time dependence of the fluorescence anisotropy can be predicted from the differences in fluorescence lifetimes and residual anisotropies of the probes located in different sites. Using this model, the local fluorescence anisotropy parameters and the relative contributions of the lipid probe octadecyl rhodamine B in a lipid environment and in the vicinity of bacteriophage M13 coat protein reconstituted in phospholipid bilayers, composed of 80% 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 20% 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol have been determined experimentally. At 40°C, the correlation times for bound and free probes are 2.3 and 3.0 ns, respectively, while the corresponding order parameters are 0.85 and 0.62, respectively.Abbreviations ESR electron spin resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol - L/P ratio phospholipid to coat protein molar ratio - <> average fluorescence lifetime - r(0) initial anisotropy - r() residual anisotropy On leave of Shanghai Medical Equipment Research Institute, 77 Jiang Ning Rd. Shanghai, People's Republic of China Offprint requests to: M. A. Hemminga