O2-triggered changes of membrane fatty acid composition have no effect on Arrhenius discontinuities of respiration in sycamore (Acer pseudoplatanus L.) cells

Sycamore cells (Acer pseudoplatanus L.) in suspension culture were grown at 25 degrees C in culture medium containing two oxygen concentrations: 250 microM O2 (standard conditions) and 10 microM O2 (O2-limiting conditions). The decrease of O2 concentration in the culture medium did not modify significantly the relative proportion of each phospholipid. In contrast, the molar proportion of fatty acids was dramatically changed in all lipid classes of the cell membranes; the average percentage of oleate increased from 3 to 45% whereas that of linoleate decreased from 49 to 22%. When normal culture conditions were restored (250 microM O2), oleate underwent a rapid desaturation process; the loss of oleic acid was associated with a stoichiometric appearance of linoleic acid at a rate of about 4 nmol of oleate desaturated/h/10(6) cells. Under these conditions, no change in the Arrhenius-type plots of the rate of sycamore cell respiration was observed; the values of the transition temperature and of the Arrhenius activation energy (Ea) associated with the cell respiration as well as with the respiration-associated enzymes remained unchanged. Thus it was concluded that the fact that a strong decrease in the fraction of unsaturated fatty acid residues present in the mitochondria had no effect on electron transport rates and Arrhenius plot discontinuities casts doubt on the significance of such changes in terms of chilling injury. Finally it is suggested that some of the Arrhenius discontinuities observed at the level of membrane enzyme could be the consequence of intrinsic thermotropic changes in protein arrangement independent of lipid fluidity.     

Effects of ATP and cyclic AMP on the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes

As turkey erythrocytes were progressively depleted of ATP by preincubation with dinitrophenol, the (Na+ + K+ + 2Cl-)-cotransport system (assayed by the bumetanide-sensitive fraction of 86Rb+ influx) became less responsive to activation. The dependence upon intracellular ATP concentration was significantly steeper for transport activated by hypertonic shock (halfmaximal activity at 0.7 mM ATP) than for that activated by either epinephrine or cyclic AMP (halfmaximal activity at 1.7 mM ATP). Upon removal of epinephrine or cyclic AMP from cells that had been preincubated with those substances, bumetanide-sensitive transport activity declined sharply, even though the intracellular cyclic AMP concentration was still over 10-fold that required to maximally activate the transport system. These data are in agreement with the notion that the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by cyclic AMP, presumably through the 'classical' pathway involving a protein kinase. They do however indicate that some other, as yet undefined aspect of cyclic AMP metabolism is important for the maintenance of transport activity.     

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered ‘cryptic species’. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex‐linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent‐based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp ), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp ). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.     

Ultrastructural visualization of the internalization of low density lipoprotein by human placental cells

Low density lipoproteins (LDL) were conjugated to colloidal gold to visualize the route for internalization of LDL in the cultured cells of human term placenta. Cells were obtained from placental villi (caesarian section) by a standard trypsin-DNase dispersion method followed in some cases by a Percoll gradient centrifugation step. Employing electron microscopy it was observed that after 3 days of culture, cells obtained by trypsin-DNase dispersion were a mixture of macrophages, mononucleated cells and large multinucleated cells. When the cells were incubated for 3 days after the Percoll purification, essentially multinucleated cells identical to the syncytiotrophoblast were present. The number of LDL receptor was increased by preincubation in medium with lipoprotein depleted serum. In binding experiments cells incubated at 4 degrees C for 2 h with medium containing gold LDL conjugates showed gold LDL attached to the plasma membrane without characteristic localization. After incubation with gold LDL at 37 degrees C for various times, the three cellular types showed ligand internalization. Gold LDL endocytosis involved first coated pits but also uncoated plasmalemmal invaginations. Then gold LDL was further observed in coated and non coated vesicles, smooth walled endosomes, multivesicular bodies and tubular vesicles. Lastly free gold particles were observed in lysosome like dense bodies. These results prove the internalization of gold LDL conjugates by human cultured placental cells, particularly by syncytiotrophoblast like multinucleated cells. This accumulation of LDL (the major cholesterol carrying protein in humans) is recognised to be responsible for the exogenous cholesterol supply indispensable to the progesterone biosynthesis and cellular growth of the placenta.     

Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts

Mesophyll derived protoplasts ofCatharanthus roseus cv. Little pinkie were fused with protoplasts derived from an habituated cell line ofC. roseus. Polyethylene glycol was used as agglutinating agent while fusions were induced by square pulses. Best results were obtained by fusing protoplasts from primary leaves with those from three-day-old cell cultures. Adding calcium ions considerably enhanced heterofusion rate. Good cell viabilities indicated that this fusion process was not cytotoxic. The heterofusion frequency was up to 10% or more. Most of the heterokaryons were able to regenerate their cell walls and underwent division. Communicated by J. TUPY