Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.     

Expression of cFABP and PPAR in trophoblast cells: effect of PPAR ligands on linoleic acid uptake and differentiation

Throughout gestation, fetal growth depends, in part, on placental transfer of maternal essential fatty acid (EFA) and long-chain polyunsaturated fatty acid. All fatty acid (FA) can cross lipid bilayer by simple diffusion, such as those in the syncytiotrophoblasts, the multinucleated, terminally differentiated trophoblast cells. The trophoblasts differentiation process is accompanied by an increase of human chorionic gonadotropin (hCG) secretion and an inhibition of Human Achaete-Scute Homologue-2 expression (Hash-2). Furthermore, a number of FA-binding proteins (FABPs) have been identified in membrane and cytoplasm of mammalian cells, which are thought to facilitate the transfer of FA across membranes and their intracellular channeling. Thus, the aim of this study was to investigate the implication of cFABPs in linoleic acid (LA) uptake by human trophoblast cells according to differentiation. Moreover, since peroxisome proliferator-activated receptor (PPARs) regulate the expression of cFABP and play an important role in trophoblast cells differentiation, the effects of PPARs ligands are verified on cFABP expression and differentiation. Herein, we reported the increase of the expression of liver and heart FABP (L- and H-FABP) upon differentiation of trophoblast cells, an inhibition of PPAR alpha and beta, while PPAR gamma levels remains unchanged. The nonselective peroxisome-proliferating agents, bezafibrate and LA, impaired trophoblast differentiation, and reduced L- and H-FABP expression. Furthermore, cobalt, a chemical agent known to mimic hypoxia, inhibits trophoblast cells differentiation and diminishes H-, L-FABP and PPARs expression. Finally, both treatments show no influence on LA uptake by trophoblast cells. In conclusion, this study showed that there is no correlation between the expression of H- and L-FABP and LA uptake by trophoblast cells and that bezafibrate and LA greatly impaired trophoblast cells differentiation.     

New antibacterial peptide derived from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.     

tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase. Assay development and characterization of the recombinant enzyme

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ). The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme. Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA(Tyr) in an E. coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and [U-ribosyl-(14)C]AdoMet was synthesized. The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate. Unexpectedly, the enzyme was inhibited by Mg(2+) and Mn(2+) in millimolar concentrations. The steady-state kinetic parameters were determined to be K(m)(AdoMet) = 101.4 microm, K(m)(tRNA) = 1.5 microm, and k(cat) = 2.5 min(-1). A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K(m)(RNA) = 37.7 microm and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.     

Studies on two species of Asellus in Rutland Water

The study comprised a comparison of the ecology of two species of Asellus (A. aquaticus and A. meridianus), and had two main aims. First the energy budget of the population of A. aquaticus, which is one of the most abundant species in the reservoir was assessed. Secondly, the two species appear to be ecologically very similar and it has been assumed by many workers that they are in competition. The study aimed to investigate how these two species might be able to coexist.The population dynamics of both species and the interaction between them is under study, as are their relations with the other benthic animals recorded in the reservoir (see Bullock et al. 1982). The sampling programme to cover variations in depth and substratum is also described by Bullock et al. 1982. A. aquaticus occurs in both arms while A. meridianus exists only in the south arm. A preliminary examination of the available data reveals that the density of A. aquaticus has increased in the south arm while the density of A. meridianus decreased drastically in February 1980. Since then low numbers of A. meridianus have been recorded every month even in the summer (breeding) season. It is noteworthy that simultaneously the number of predators, particularly Helobdella stagnalis, increased in comparison with previous years.It was therefore decided to study the predation on Asellus in the laboratory as well as in the field by using a serological technique. Antisera were produced against each species of Asellus by injections into rabbits of precipitated proteins (antigens). Thus a positive reaction obtained from a homogenised predator when tested with the specific antiserum indicates which species of Asellus had been consumed and the percentage of predators which had fed on Asellus in every sample could be calculated. The results obtained to date reveal that considerable numbers of Asellus spp. are removed from the environment by the predators (Helobdella stagnalis, Erpobdella octoculata, Polycelis tenuis. Dendrocoelum lacteum and Dugesia lugubris).Size class structures of the population of both species were constructed to study their life cycles and to estimate the population production. Comparitive respirometry of the two species was carried out at 4 °C, 10 °C and 16 °C, using a Gilson differential respirometer. This study aimed firstly to assess one of the essential parameters in the energy budget study of A. aquaticus and secondly, since A. aquaticus appears to be more active than A. meridianus, it would be expected that a comparison of their metabolic rate at different temperature would reveal marked differences between two species. It was found that there was no significant difference between the two species at 4 °C and 10 °C, but A. aquaticus had a significantly higher metabolic rate than A. meridianus at 16 °C.Consumption and assimilation of decaying oak leaves and of Cladophora glomerata (both of which are available in the environment) by two species of Asellus are being measured at present in the laboratory at three different temperatures (4 °C, 10 °C and 16 °C), and will contribute to the estimation of energy budgets.