Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

Thermal Rearrangement of 4-Alkyl-1,2,4-triazoles. Rearrangements in the Crystalline State

Studies of the thermolyses of 4-alkyl substituted 1,2,4-triazoles was reviewed. They were observed to rearrange at 200–350 °C to the corresponding 1-alkylated triazoles. When substituted in the 4-position with aryl- or vinylic substituents the triazoles were inert to thermolysis, contrary to what was observed for the 4-alkyl- and 4-allyl substituted systems. The mechanisms for the reactions were elucidated, e.g., by studies of substituents effects and by kinetic measurements in solution as well as for the neat samples. Reactions in solutions were slow. The rearrangements in melts of the neat triazoles readily proceeded to the products, and were proposed to take place via a series of nucleophilic displacement steps. X-ray crystallographic measurements of selected structures, showed that the interatomic distances and angles between the relevant atoms in these structures, to a large degree resembled the geometry expected for the S_N2-type transition states proposed for the rearrangement mechanism. Thus, thermolyses of a series of triazole structures at temperatures below their melting points, confirmed that rearrangements actually did take place. The “kinetics” of the reactions in the crystalline state were investigated and rate constants and thermodynamic data were correlated with the structural characteristics of the crystals.     

Influence of the nontarget mollusc Marisa cornuarietis on the hourly cercarial production of Schistosoma mansoni from Biomphalaria glabrata

A comparative study of hourly cercarial productivities of Schistosoma mansoni from infected Biomphalaria glabrata was carried out in the presence of either healthy B. glabrata (control) or healthy Marisa cornuarietis (experimental). The results showed that, with M. cornuarietis, almost all the hourly cercarial productivities increased by a factor varying from 1.3 to 2.5 without modification of the shedding period.     

Alginate as immobilization material: III. Diffusional properties

The rate of diffusion of serum albumin (MW 6.9 x 10(4) D) out of beads of calcium alginate gels depends upon the concentration and uronic acid composition of the alginate (ManA/GulA ratio), the conditions under which the beads are produced, the pH, and the temperature. The diffusion coefficient decreases with increasing alginate concentration, and (ManA/GulA) ratio and with decreasing pH. Diffusion out of the beads, in which the alginate is uniformly distributed (homogeneous gel), is faster than out of the beads in which the alginate is concentrated at the surface (inhomogeneous gel). The temperature dependence of the diffusion coefficient follows the Arrhenius law, with an activation energy of approximately 23 kJ x mol(-1).     

Gene flow inferred from seed dispersal and pollinator behaviour compared to DNA analysis of restriction site variation in a patchy population of Lotus corniculatus L.

Summary Gene flow was investigated in a natural population of Lotus corniculatus L. (Fabaceae) using a combination of pollen and seed dispersal studies and a recombinant DNA technique. The population is spatially heterogeneous and grows with Empetrum nigrum. L. corniculatus is pollinated by the pollen-collecting bumblebee Bombus lapidarius L. Most pollinator flights occurred within patches, as bees usually visit nearest-neighbour plants, show no marked directionality, and forage mostly within patches. Gene flow by seeds is also limited, reinforcing the pattern of gene flow within patches. However, 2.6% of pollinator flights are between patches and considerable pollen carryover also occurs. Thus, gene flow between patches is potentially sufficient to retard or prevent genetic differentiation in spite of the patchy sub-structuring of the population. A sub-set of the population was analysed for restriction fragment length polymorphisms (RFLPs) to document the actual gene flow pattern of the population. The DNA analysis revealed significant levels of genetic differentiation between the patches. The level of gene flow that can be inferred from the distribution of genetic variation is surprisingly restricted, as compared to gene flow inferred from pollinator behaviour, and emphasizes that stochastic processes like genetic drift and founder effects may have a strong impact on the prevailing genetic structure.     

Influence of three vesicular-arbuscular mycorrhizal fungi (Glomaceae) on the activity of specific enzymes in the root system of Cucumis sativus L.

The influence of three vesicular-arbuscular mycorrhizal (VAM) Glomus species on the activity of enzymes in the roots of Cucumis sativus was tested. Cucumber plants were grown in a split-root system, in which colonized and uncolonized roots of a single plant could be separated. The activity of the host root malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (Gd), glutamate oxaloacetate transaminase (GOT) and glutamate dehydrogenase (GDH) was measured on a densitometer after separation of the host and fungal enzymes on polyacrylamide gels.The results showed that only minor changes in the activity of the host root enzymes occurred after VAM inoculation. Gd was stimulated by VAM and phosphorus, and one of the fungi decreased the activity of GDH in the host plant when both parts of the root system were colonized.     

Microbial biomass and activity in subsurface sediments from Vejen,Denmark

Subsurface sediment samples were collected from 4 to 31 m below landsurface in glacio-fluvial sediments from the Quaternary period. The samples were described in terms of pH, electrical conductivity, chloride concentration, organic matter content, and grain size distribution. Viable counts of bacteria varied from 0.5 to 1,203 x 103 colony forming units/g dry weight (gdw); total numbers of bacteria acridine orange direct counts (AODC) varied from 1.7 to 147 × 10⁷ cells/gdw; growth rates (incorporation of [³H]-thymidine) varied from 1.4 to 60.7 × 10⁴ cells/(gdw · day); and rate constants for mineralization of ¹⁴C-labelled compounds varied from 0.2 to 2.3 × 10^–3 ml/(dpm · day) for acetate, and from 0 to 2.0 × 10^–3 ml/(dpm · day) for phenol. Sediment texture influenced the total number of bacteria and potential for mineralization; with increasing content of clay and silt and decreasing content of sand, AODC increased and the mineralization rate declined. Intrinsic permeability calculated from grain size correlated positively with mineralization rate for acetate. Statistical correlation analysis showed high correlations between some of the abiotic parameters, but it was not possible to point out a single abiotic parameter that could explain the variation of size and activity of the microbial population. The microbial data obtained in these geologically young sediments were compared to literature data from older sediments, and this comparison showed that age and type of geological formation might be important for the size and activity of the microbial populations.Offprint requests to: H.-J. Albrechtsen.     

Factors controlling nitrification and denitrification: A laboratory study with gel-stabilized liquid cattle manure

Nitrification and denitrification were studied in a millimeterscale microenvironment using a two-phase system with a liquid manure-saturated layer. Samples consisted of liquid cattle manure and air-dried soil stabilized with silica gel, placed between two aerobic soil phases with a water content near field capacity. A high potential for NH₄ ⁺ oxidation developed within 0–2 mm distance from the interface, and NH₄ ⁺ diffused only 10–20 mm into the soil. Some NH₄ ⁺ was probably immobilized by microorganisms in the soil between 0 and 4 days, after which nitrification was the only sink for NH₄ ⁺. A potential for denitrification developed within the manure-saturated zone. Maximum rates of both potential and actual denitrification were recorded by Day 4, but denitrification continued for at least 2–3 weeks. The potential for nitrification peaked after 14 days. When the pH of the manure was adjusted to 5.5, nitrification was reduced close to the interface, and NH₄ ⁺ penetrated further into the soil before it was oxidized. The pH adjustment had an inhibitory effect on denitrification: Both potential and actual rates of denitrification were almost eliminated for several days. The size of the manure-saturated layer strongly affected denitrification losses. With layers of 8 and 16 mm thickness, losses equivalent to 33 and 40% of the original NH₄ ⁺ pool, respectively, were estimated. When manure corresponding to a 12 mm layer was homogeneously mixed with the soil, only 0.3% was lost.Offprint requests to: S. O. Petersen.     

The human placental transferrin receptor: Reconstitution into liposomes and electron microscopy

Human transferrin receptor was isolated from Triton X-100 solubilized placental plasma membranes by a rapid one-step chromatographic procedure based on immunoadsorption of the receptortransferrin complex on anti-transferrin Sepharose and lectin-affinity on wheat germ agglutinin. Following exchange of Triton X-100 with CHAPS or n-octylglucoside, the purified receptor was incorporated into egg phosphatidylcholine liposomes upon, detergent removal by dialysis (lipid/protein ratio 15:1 to 45:1 (w/w) Reconstitution of the receptor was confirmed by trypsin cleavage to dissociate the large extracellular receptor domain from the liposomal membranes. Electron micrographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed ographs of the receptor-lipid recombinants negatively stained with sodium sillicotungstate, showed that the receptor molecules distributed very inhomogeneously on the liposomes, most receptors being clustered. Single copies of the receptor were seen as elongate structures (5×10 nm) oriented with their long axis parallel to the liposome surface and separated from this by a 2–3 nm gap. This result provides evidence for a narrow connecting link between the globular extracellular receptor domain and the membrane spanning segment.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - PAGE polyacrylaminde gel electrophoresis - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - WGA wheat germ agglutinin