Flux control at the ecosystem level

The analysis of metabolic control has reached a high level of understanding of the regulation in cellular metabolic pathways. However, as soon as we leave the realm of cell compartments and enter into the demise of coordination at the organism or ecosystem level, control theory enters unstable ground. Organisms act as individuals. Here, I compare control features at different levels of organization (cell to ecosystem), to indicate how we may approach understanding of control in complex and multiple-species systems.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Response to Gravity by Zea mays Seedlings : I. Time Course of the Response

Gravistimulation induces an asymmetric distribution of free indole-3-acetic acid (IAA) in the cortex-epidermis of the Zea mays L. cv `Stowells Evergreen' mesocotyl within 15 minutes, the shortest time tested. IAA was measured by an isotope dilution method as the pentaflurobenzyl ester. The per cent IAA in the lower half of the mesocotyl cortex was 56 to 57% at 15, 30, and 90 minutes after stimulus initiation. Curvature is detectable in the mesocotyl within 3 minutes after beginning gravitropic stimulation. The rate of curvature of the mesocotyl increases during the first 60 minutes to a maximum of about 30° per hour. Thus, the growth asymmetry continues to increase for 45 minutes after hormone asymmetry is established.

Free IAA occurs predominantly in the stele of the mesocotyl whereas esterified IAA is mainly in the mesocotyl cortex-epidermis. This compartmentation may permit determining in which tissue the hormone asymmetry arises. Current data suggest the asymmetry originated in the stele.

     

Continuous measurement of minute ventilation and gaseous metabolism of newborn infants

A system of instrumentation for continuous measurement of gaseous metabolism and minute volume (VI) in the human newborn is described. O2 uptake and CO2 production are measured by open-circuit techniques utilizing a Servomex OA184 differential paramagnetic O2 analyzer and a BEckman LB-2 infrared CO2 analyzer. VI is measured with bias-flow pneumotachometry. Bench performance is described, methodological errors are defined, and clinical data are presented. The instrumentation is capable of safe, accurate, and continuous measurement of respiratory and metabolic variables in low-birth-weight infants.     

Ecophysiological investigations on lichens of the Negev desert

Summary Previous publications have reported on investigations of CO₂ exchange in the desert lichenRamalina maciformis both in its natural habitat in the Negev and in the laboratory. Utilizing laboratory data, net photosynthesis and dark respiration were expressed as mathematical functions of the most important environmental factors. Based on these relationships, a model is developed that allows one to predict CO₂ exchange of the plant. Input data are light intensity, temperature, and water content of the thallus, together with a measure of the rate of the seasonal change of photosynthetic and respiratory activity. The validity of the model is tested by comparing simulated daily courses of CO₂ uptake and release of the lichen with independent results of CO₂ exchange measurements conducted in the field during and after the condensation of dew. The sensitivity of the model is shown by simulating changes in the input data of temperature and water content of the lichen.This paper is dedicated to Dr. h.c. Oscar Klement on the occasion of his 80th birthday     

Inactivation and stabilization of stabilisins in neat organic solvents

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.     

Incorporation of phosphonic acid diesters into lipid model membranes. Part II. X-ray and neutron diffraction studies.

Mixtures of egg phosphatidylcholine and phosphonic acid diethyl or dibutyl esters of the general type RP(O)(OR')2 with R = hexane or dodecane were studied at room temperature in the fluid lamellar state by X-ray and by neutron diffraction. Generally a molar ratio of lipid and ester of 1:0.5 was used. Additionally an equimolar lipid/ester mixture of hexane phosphonic acid diethyl ester was studied. Depending on the ester used and its concentration a single L alpha-phase was observed above a certain water content which changes to an L alpha + water two phase system at high water concentration. Despite the large amounts of the amphiphilic ester molecules incorporated in the membrane and their high molecular asymmetry, the mixtures qualitatively show the typical hydration and swelling behaviour of non-charged lipid membranes. However, the incorporation of the esters induces a higher hydration capacity, a lateral extension and a decrease in membrane thickness. The position of the ester molecules and their orientation in the membrane were determined by neutron diffraction using partially deuterated esters. The esters were found to be located with their phosphonic moiety near or in the lipid/water interface. The lamellar structure contradicts this location of the cone-shaped ester molecules which should increase the tendency to form hexagonal structures. However, the experimental findings can be understood if one considers a partial interdigitation of the last hydrocarbon groups of the lipid chains accompanied by a larger disorder in the hydrophobic centre of the membrane. In the case of hexane phosphonic acid dibutyl ester, a vertical translocation of the ester takes place below a certain water content where it is distributed between two locations at the lipid water interface and the centre of the membrane.     

Site-directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Binding of the peripheral components E1p and E3.

Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex. Upon binding of peripheral components, the 24-subunit core of A. vinelandii wild-type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers. Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (delta P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli. Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes. It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.     

Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [L?bermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.