Comments on “Seed germination as a thermobiological problem”

Summary In the above mentioned article [1] the notion of a time-dependent Gibbs free energy has been introduced to explain the observed time-pattern of embryo growth in seeds. Furthermore, the notion of a non-random thermal communication has been inferred from an inspection of the shapes of germination time distributions. It can be shown, however, that the reasoning leading to the time-dependent thermodynamic potential is based on inappropriate interpretation of kinetic equations, and that the shape of the distributions of germination time might be a natural consequence of the initial distribution of embryo size in the seeds.Discussion about the Paper Seed Germination as a Thermobiological Problem by L. G. Labouriau Radiation and Environmental Biophysics 15:345–366 (1978)     

Vegetation of a snow bed at Godhavn, West Greenland

The vegetation of a snow bed has been described by a pin-point method and a modified Raunkiær frequency analysis. The thawing of the snow has been followed and some soil properties have been investigated. It is concluded that the composition of the vegetation in the snow bed is influenced mainly by the duration of the growth period, but locally the density and the species composition are determined by the downward flow of the melt water.     

Dialysis Continuous Process for Ammonium-Lactate Fermentation of Whey: Mathematical Model and Computer Simulation

A mathematical model was developed to describe a dialysis process for the continuous fermentation of whey lactose to lactic acid, with neutralization to a constant pH by ammonia. In the process, whey of a relatively high concentration is fed into the fermentor circuit at a relatively low rate so that the residual concentration of lactose is low. The fermentor effluent contains ammonium lactate, bacterial cells, and residual whey solids and could be used as a nitrogen-enriched feedstuff for ruminant animals. Only water is fed into the dialysate circuit at a relatively high rate. The dialysate effluent contains purified ammonium lactate and could be converted to lactic acid and ammonium sulfate for industry. The fermentation was specifically modeled as a set of equations representing material balances and rate relationships in the two circuits. Dialysis continuous fermentations, in general, were modeled by combining these equations and by using dimensionless parameters. The generalized model was then solved for the steady state and used to simulate the specific fermentation on a digital computer. The results showed the effects of various material and operational and kinetic parameters on the process and predicted that it could be operated efficiently.     

Interaction of invertase with polyelectrolytes

In connection with our work on polyelectrolyte complex formation with polyampholytes, the interaction between invertase and several linear polyelectorlytes has been investigated by means of turbidimetry, light scattering measurements, and determination of the enzyme activity. Polyelectrolyte complex formation of invertase was shown to occur with cationic polyelectrolytes only. The light-scattering data yield information on aggregation and desegregation processes in complex formation. As indicated by our results, only a part of the protein molecules is engaged in this Coulombic interaction, and this part shows a rather small enzyme activity only. Thus, a direct interaction between invertase and a cationic polyelectrolyte is no effective approach to enzyme binding, but a complete immobilization of invertase can be achieved via an "inclusion flocculation" with a symplex formed by interaction between an anionic and a cationic linear polyelectrolyte or via immobilization in symplex microcapsules.     

Isolation of salmonellas by immunomagnetic separation.

Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10(7] were generally incubated with 10(4) Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.0 +/- 7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9 +/- 12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salm. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors.

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.     

Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and microglia.

The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system.     

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.     

Non-covalent interactions result in aggregation of surface antigens of the parasitic nematode Trichinella spiralis.

Surface antigens of three stages of the nematode worm Trichinella spiralis has been labelled with iodine and examined by sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. A variety of interactions were defined: the infective larva surface antigens formed a spectrum of aggregates from 50kDa to greater than 1000kDa from subunits of 47kDa and 90kDa; in the adult worms of 60kDa complex arose fron interaction between two dissimilar molecules of 40kDa and 20kDa; the new-born larvae components formed homologous dimers from a 58kDa molecule. Aggregating molecules were adherent to lentil lectin-Sepharose and are therefore glycoproteins. The interactions observed were completely abolished by boiling in SDS/mercaptoethanol, but only partially destroyed by boiling in SDS/iodoacetamide. Based upon this, the associations can be characterized as non-covalent, but disulphide-bond-dependent. It is suggested, but not proved, that the aggregates arise from strong non-covalent hydrophobic interaction sites which are stabilized by intrachain disulphide bonds in the molecules concerned.