Principles and pitfalls in designing site-directed peptide ligands

An immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X-ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallo-graphically refined structures of 14 peptide–protein complexes at 2.7 Å resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change inconformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program anend-to-end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligandswere diffused into preexisting crystals. In sucha large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints. © 1993 Wiley-Liss, Inc.     

Citric acid and polyols production by Aspergillus niger at high glucose concentration in solid state fermentation on inert support

Summary Aspergillus niger cultures at high initial glucose concentration (up to 400 g/1) on Amberlite as inert support were carried out. Citric acid was accumulated in the support showing high concentration (94.54 g/l) and productivity (1.35 g/l h) without inhibition related to the presence of metals (Mn²⁺, Zn²⁺, Co²⁺, Cu²⁺, and Ca²⁺) at high concentrations. Citric acid accumulation was clearly associated with both, glycerol production and to the age of the culture. Glycerol and erythritol, the major osmoregulator metabolites, were also produced (8.16 and 24.57 g/l respectively) at 400 g/l of glucose.     

Documentation of the presence of Gmelinoides fasciatus (Stebbing, 1899) and the native benthic fauna in the Volga River at Tver (Tver Region,Russia)

In the Volga Basin, the small Baikalian amphipod Gmelinoides fasciatus was introduced in 1965 into the Gorky reservoir in order to enhance fish production; it appeared in 1986 in the Rybinsk reservoir and we recorded it during monitoring activities in 2006 at Tver. In total, at the monitoring site Tver/Migalovo 69 benthic invertebrate taxa were identified. We compared data from three summer seasons. During summer low flow period Gmelinoides fasciatus did not exceed a share of 12.6% considering individual (ind) densities (mean abundance 165 ± 104 ind m^?2) and 14.2% considering biomass (mean biomass 0.39 ± 0.44 g m^?2). Abundances and biomass of G. fasciatus were shown to be stable over three years and no increase was observed. The monthly dataset (March–November 2008) revealed dynamics in relation to the native benthic communities and it was shown that the maximal densities of Gmelinoides did not exceed 587 ind m^–2. Understanding the effects on benthic communities caused by the invasive amphipod Gmelinoides fasciatus is crucial in order to predict further developments in European inland waters and to establish management strategies.     

Disputed Areas and the 10-Year Time Frame: A Legal Lacuna?

Where a coastal state intends to delineate, in accordance with Article 76 of the 1982 Law of the Sea Convention, the outer limits of its continental shelf beyond 200 nautical miles, it is to submit scientific and technical data of such proposed limits to the Commission on the Limits of the Continental Shelf within 10 years of the entry into force of the Convention for that state. This obligation has subsequently been subject to a general de facto amendment by which the commencement of the 10-year time frame has been postponed to May 13, 1999, for the states for which the Convention had entered into force prior to the above date. This article discusses whether any provision in the Convention, any subsequent agreement, or any subsequent practice render inoperative the 10-year time frame with regard to those parts of the outer continental shelf that are disputed areas. It is concluded that nothing in the Convention, no subsequent agreements, and no subsequent practice allow for derogation of the 10-year time frame with regard to disputed areas.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

Phenotypic diversification of a cultured tumor line as a function of substratum

We have found that a murine hepatoma displays a considerable phenotypic diversification in culture, which depends upon the substratum utilized, and is manifested by the formation of multicellular structures of differing geometry: Monolayer on glass and plastic, thick multilayer pads on Gelfilm, and spheroids on agar and agarose. These multicellular morphological phenotypes were assayed without disruption to ascertain their antigenicity in vitro and their tumorigenicity in vivo and to obtain quantitative information on the effect of the spatial arrangement of the hepatoma cells upon the ability of each multicellular structure to interact, as a whole, with molecules and cells in its surroundings. The antigenicity of the multicellular structures was determined with calibrated probes and a methodology that measures the total antigenicity, as well as antigenicity per unit of surface area. Antigenicity was found to differ in the following decreasing order: Monolayer on plastic > spheroids on agarose > spheroids on agar > multilayer on Gelfilm. At least part of these antigenic variants arise from different degrees of masking of the structures' surface determinants by a trypsin-sensitive material. The multicellular phenotypes also differed in tumorigenicity. When assayed in syngeneic hosts under comparable conditions, agar-grown spheroids produced the fewest tumors, whereas Gelfilm-grown multilayers produced the most. These two independent sets of data show that the various geometries that a tumor tissue is induced to acquire by the culture substratum are accompanied by a distinctive combination of surface and biological properties.     

Circadian communication between unicells?

Populations ofGonyaulax polyedra, in two different phases, about 11 h apart, were mixed, and the intensity of their spontaneous bioluminescence glow recorded for about 2 wk under conditions of constant dim (35±3 μE/m²/s) white light and constant temperature (19.0±0.3°C). The phases and amplitudes of glow signals recorded from mixed cultures were compared with those obtained from the arithmetic sum of the intensity data from two control vials. Peaks in control cultures generally remained separate, but there was a spontaneous increase in the period beginning 6–11 d after the onset of constant conditions. This did not occur in cultures in which the medium was exchanged with fresh medium every 2 d. In the actual mixes of two cultures there was a merging of the two subpeaks in the signal, which did not occur when the medium was exchanged. The results indicate that conditioning of the medium by cells may affect the period of the circadian rhythm and that this might result in a type of communication. Supported by the Deutsche Forschungsgemeinschaft; present address     

Biotic and abiotic determinants of the formation of ant mosaics in primary Neotropical rainforests

1. Ants are widespread in tropical rainforests, including in the canopy where territorially dominant arboreal species represent the main part of the arthropod biomass. 2. By mapping the territories of dominant arboreal ant species and using a null model analysis and a pairwise approach this study was able to show the presence of an ant mosaic on the upper canopy of a primary Neotropical rainforest (c. 1 ha sampled; 157 tall trees from 28 families). Although Neotropical rainforest canopies are frequently irregular, with tree crowns at different heights breaking the continuity of the territories of dominant ants, the latter are preserved via underground galleries or trails laid on the ground. 3. The distribution of the trees influences the structure of the ant mosaic, something related to the attractiveness of tree taxa for certain arboreal ant species rather than others. 4. Small‐scale natural disturbances, most likely strong winds in the area studied (presence of canopy gaps), play a role by favouring the presence of two ant species typical of secondary formations: Camponotus femoratus and Crematogaster levior, which live in parabiosis (i.e. share territories and nests but lodge in different cavities) and build conspicuous ant gardens. In addition, pioneer Cecropia myrmecophytic trees were recorded.     

Effect of dolichyl monophosphate on the permeability properties of lipid membranes

The effect of dolichyl monophosphate on the permeability properties of dimyristoylphosphatidylcholine bilayers to alkaline cations, Ca²⁺ and glucose has been determined by stop-flow spectrophotometry. The results show that, in con trast to free dolichol effects, the monophosphate derivative increased the permeability following a decreasing order of the permeating particle size. Phase diagrams indicate that dolichyl monophosphate is fully incorporated into the phosphatidylcholine bilayer around 0.75% weight/weight ratio. For these ratios, the permeation of ions is higher in the gel than in the liquid crystalline state.