Degradation of Escherichia coli chromosomal and plasmid DNA in serum

Incubation of serum-sensitive [3H]thymidine labelled Escherichia coli PC2166 (RSF1030) and E. coli AM1281 (pBR322) harbouring small plasmids (mol. wt 5.5 X 10(6) and 2.6 X 10(6] in serum resulted in killing of 99.9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation. The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria. Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum. However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum.     

Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties.     

Qualitative and Quantitative Interactions between Microphytobenthos and Herbivorous Meiofauna on a Brackish Intertidal Mudflat

Micro- and meiofauna are the predominant consumers of diatoms on a brackish intertidal mudflat. The impact of grazing on the benthic diatom populations was studied by field observations and feeding experiments on a few abundant members of the community. Only small fractions of the microphytobenthic biomass and production are converted by herbivores. A hypothesis is presented explaining the growth kinetics and productivity of diatom populations and the inefficient transfer of carbon into herbivore foodchains. Data on feeding rate and population dynamics of the nematode species, Eudiplogaster pararmatus, are discussed in view of the seasonal succession of edible diatom species.     

Drying Increases Intracellular Partitioning of Amphiphilic Substances into the Lipid Phase : Impact on Membrane Permeability and Significance for Desiccation Tolerance

Previously we proposed that endogenous amphiphilic substances may partition from the aqueous cytoplasm into the lipid phase during dehydration of desiccation-tolerant organ(ism)s and vice versa during rehydration. Their perturbing presence in membranes could thus explain the transient leakage from imbibing organisms. To study the mechanism of this phenomenon, amphiphilic nitroxide spin probes were introduced into the pollen of a model organism, Typha latifolia, and their partitioning behavior during dehydration and rehydration was analyzed by electron paramagnetic resonance spectroscopy. In hydrated pollen the spin probes mainly occurred in the aqueous phase; during dehydration, however, the amphiphilic spin probes partitioned into the lipid phase and had disappeared from the aqueous phase below 0.4 g water g⁻¹ dry weight. During rehydration the probes reappeared in the aqueous phase above 0.4 g water g⁻¹ dry weight. The partitioning back into the cytoplasm coincided with the decrease of the initially high plasma membrane permeability. A charged polar spin probe was trapped in the cytoplasm during drying. Liposome experiments showed that partitioning of an amphiphilic spin probe into the bilayer during dehydration caused transient leakage during rehydration. This was also observed with endogenous amphipaths that were extracted from pollen, implying similar partitioning behavior. In view of the fluidizing effect on membranes and the antioxidant properties of many endogenous amphipaths, we suggest that partitioning with drying may be pivotal to desiccation tolerance, despite the risk of imbibitional leakage.     

Dehydration-induced conformational changes of poly-l-lysine as influenced by drying rate and carbohydrates

The conformation of hydrated and air-dried poly-l-lysine in thin films was studied using Fourier transform IR spectroscopy in the amide-I region. Hydrated poly-l-lysine has a random coil conformation. Upon slow drying of small droplets of the polypeptide solution over a period of several hours, an extended β-sheet conformation is adopted. This conformational transition can be prevented by fast air-drying within 2–3 min. Slow air-drying in the presence of sucrose also preserves the aqueous conformation and results in the formation of a glassy state. Comparison of shifts of the OH band with temperature indicates that sucrose/poly-l-lysine mixtures form a molecularly more densely packed glassy matrix, having a higher glass transition temperature (T_g), than sucrose alone. Whether direct interaction of sugar and polypeptide or glass formation is involved in the stabilization during slow air-drying was studied by drying in the presence of glucose or dextran. Compared with dextran (and sucrose to a lesser extent), glucose gives superior protection. Dried glucose has the lowest T_g and the best interacting properties. We conclude that either immobilization by fast air-drying or sufficient interaction with a protectant through hydrogen bonding (slow drying) plays the leading role in the preservation of the aqueous protein structure.     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture.

We studied the effect of fetal calf serum and serum proteins fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296--307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine beta-globulins reduced this transfer, although to a lesser extent than whole serum. alpha-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A gamma-globulin fraction failed to exhibit any effect on the uptake of [14C]phosphatidylcholine by the cells. All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles reincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine ot parenchymal cells. These observation were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of tm proteins. On the other hand, we cannot rule out that plasma membrane modifications are involved in the mechanism of inhibition as well.