Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by¹H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (P^k-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tj^a serum is the placenta tissue, resulting in termination of the pregnancy.     

Bromouracil mutagenesis in Escherichia coli evidence for involvement of mismatch repair.

Summary Bromouracil mutagenesis was studied in several strains of E. coli in combination with measurement of incorporation of bromouracil in DNA. For levels below 10% total replacement of bromouracil for thymine, mutagenesis was negligible compared with higher levels of incorporation. Such a nonlinear response occurred both when the bromouracil was evenly distributed over the genome and when a small proportion of the genome was highly substituted. Also, the mutation frequency could be drastically lowered by amino acid starvation following bromouracil incorporation. These observations suggest the involvement of repair phenomena. Studies of mutagenesis in recA ^– and uvrA ^– mutants, as well as studies of prophage induction, did not support an error prone repair pathway of mutagenesis. On the other hand, uvrD ^– and uvrE ^– mutants, which are deficient in DNA mismatch repair, had much increased mutation frequencies compared with wild type cells. The mutagenic action of bromouracil showed specificity under the conditions used, as demonstrated by the inability of bromouracil to revert an ochre codon that was easily revertable by ultraviolet light irradiation. The results are consistent with a mechanism of bromouracil mutagenesis involving mispairing, but suggest that the final mutation frequencies depend on repair that removes mismatched bases.     

Radiation-induced heat-labile sites that convert into DNA double-strand breaks

The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.     

A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC–MS/MS

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of adducts from acrylamide, glycidamide and ethylene oxide to N-terminal valines in hemoglobin (Hb) was developed. This new procedure is based on the same principles as the N-alkyl Edman procedure for analysis of adducts from electrophilic agents to N-terminal valines in Hb. The N-substituted valines can be detached, enriched and measured selectively as thiohydantoins by the use of an Edman reagent, in this case fluorescein isothiocyanate (FITC). This procedure is denoted as the “adduct FIRE procedure” as the FITC reagent is used for measurement of adducts (R) formed from electrophilic compounds with a modified Edman procedure. In this study, fluorescein thiohydantoin (FTH) analytes of N-substituted valines from acrylamide, glycidamide and ethylene oxide, as well as their corresponding hepta- and tri-deuterium-substituted analogues, were synthesized. These analytes (n = 8) were then characterized by LC–MS/MS (ESI, positive ion mode) and obtained product ions were interpreted. A considerable work with optimization of the FIRE procedure™, resulted in a procedure in which low background levels of the studied adducts could be measured from 250 μL lyzed whole blood samples (human non-smokers). The analytes were enriched and purified with solid phase extraction columns and analyzed by LC–MS/MS with LOQ down to 1 pmol adduct/g Hb. Compared to other procedures for determination of N-terminal Hb adducts, the introduction of FITC has led to a simplified procedure, where whole blood also can be used, giving new opportunities and reduced hand on time with increased sample throughput.     

Dose-dependent misrejoining of radiation-induced DNA double-strand breaks in human fibroblasts: experimental and theoretical study for high- and low-LET radiation

Misrejoining of DNA double-strand breaks (DSBs) was measured in human primary fibroblasts after exposure to X rays and high-LET particles (helium, nitrogen and iron) in the dose range 10-80 Gy. To measure joining of wrong DNA ends, the integrity of a 3.2-Mbp restriction fragment was analyzed directly after exposure and after 16 h of repair incubation. It was found that the misrejoining frequency for X rays was nonlinearly related to dose, with less probability of misrejoining at low doses than at high doses. The dose dependence for the high-LET particles, on the other hand, was closer to being linear, with misrejoining frequencies higher than for X rays, particularly at the lower doses. These experimental results were simulated with a Monte Carlo approach that includes a cell nucleus model with all 46 chromosomes present, combined with realistic track structure simulations to calculate the geometrical positions of all DSBs induced for each dose. The model assumes that the main determinant for misrejoining probability is the distance between two simultaneously present DSBs. With a Gaussian interaction probability function with distance, it was found that the data for both low- and high-LET radiation could be fitted with an interaction distance (sigma of the Gaussian curve) of 0.25 microm. This is half the distance previously found to best fit chromosomal aberration data in human lymphocytes using the same methods (Holley et al., Radiat. Res. 158, 568-580, 2002). The discrepancy may indicate inadequacies in the chromosome model, for example insufficient chromosomal overlap, but may also be partly due to differences between fibroblasts and lymphocytes.     

Tides,tidal currents and their effects on the intertidal ecosystem of the southern bay,Inhaca Island,Mozambique

Sediment characteristics and tidal currents were studied in the 1500 ha intertidal area south of Inhaca Island,Mozambique. The tide is semi-diurnal with a range at spring of about 3 m. The area connects directly to the oceanthrough the Ponta Torres Strait and (indirectly) through several narrow tidal channels ending up in Maputo Bay.Velocities of up to 0.75 m s^-1 were measured in the Ponta Torres strait. After Low Water, Indian ocean water startsentering the tidal flats, while entrance from the Maputo Bay side is delayed by one to several hours. With onlyone tide gauge and one current meter at hand, we found indications of a higher mean sea level on the oceanic side(probably due to ocean wave set up) and a mean flow in the Ponta Torres Strait directed towards Maputo Bay. Astorm from the SW had a particularly strong influence on the sea level of Maputo Bay, and for a period of a fewdays the current flowed constantly towards Maputo Bay. Measurements in the innermost Saco channel indicatedhigher velocities during ebb than during flood. The resulting residual current will increase sedimentation in the bay,a process which is confirmed by aerial photographs and could be linked to the sanding of the coral reefs and themangrove extension in the area. Extensive sandbanks are situated around both inlets as a result of the higher currentvelocities in these areas, thereby inhibiting the settlement of finer sediments and organic matter. Where the twoflows meet, formation of mudflats and seagrass beds are favoured. Mudflats are found mainly below High WaterNeap, exposed only during spring tides. This longer inundation period favours the settlement of finer material andorganic matter. The area and location of the substrates have great influence on the richness and abundance of thebenthic fauna, as grainsize and organic matter have been correlated with richness and abundance in other studies.     

Tracing a bog-iron bloomery furnace in an adjacent lake-sediment record in Ängersjö, central Sweden,using pollen and geochemical signals

Recent studies of bloomery sites in Sweden indicate the amount of iron produced with this early low-technology smelter was greater than previously thought, which implies greater economic importance. Little is known about the history of bloomery technology, not least the timeframe over which individual bloomeries were operated, as well as their impact on the landscape because of resource consumption and pollution. In this study we performed pollen and geochemical analyses of the lake-sediment record from Rörtjärnen, which is 120 m from the remains of a documented bloomery [one radiocarbon date: ad 1300–1435 (1 σ)], in Ängersjö, Hälsingland. A surface-soil transect shows a limited geochemical signal only within 20 m of the bloomery, and the sediment pollen record provides little direct evidence of an active bloomery and is consistent with other studied sites in the area linked to forest grazing or cultivation. Instead, we find major changes in sediment geochemistry during ad 800–1200, centered on a unique peak in Pb at ad 1030–1060. These changes include, e.g., Si (biogenic) and P, together with changes in pollen (e.g., Betula, Picea, Cyperaceae), which together indicate disturbance in the forest and especially the adjoining fen. We attribute these changes to a period of bloomery-related activities predating the radiocarbon date of the charcoal from the bloomery, and suggest that date represents a late phase for the site.