Karyotypically normal and abnormal human embryonic stem cell lines derived from PGD-analyzed embryos

Although a normal karyotype is generally a requirement for stem cell lines, new applications are likely to emerge for stem cells with defined chromosomal aneuploidies. We therefore investigated the use of embryos found to be aneuploid on biopsy followed by preimplantation genetic diagnosis (PGD) with fluorescent in situ hybridization (FISH), and developmentally arrested embryos for stem cell derivation. Eleven stem cell lines were obtained from 41 embryos in 36 cultures, with higher success rate achieved from PGD-analyzed, developmentally advanced embryos (45%) than from clinically unsuitable non-PGD embryos (13%). The resulting stem cell lines were karyotyped, and surprisingly, six of the nine lines from aneuploid embryos as well as both lines from non-PGD embryos were karyotypically normal. Three lines from PGD embryos were aneuploid exhibiting trisomy 5, trisomy 16, and an isochromosome 13, respectively. None of the aneuploid lines presented the same anomally as the original PGD analysis. Our study has three important implications. First, we confirm the ability to produce stem cell lines from PGD-tested embryos as well as developmentally abnormal embryos, offering specialty stem cell lines for research into the clinically important aneuploidies. Second, we observe that stem cell derivation from apparently aneuploid embryos is often thwarted by underlying mosaicism and emerging dominance of the stem cell line by karyotypically normal cells. The corollary, however, is that regular production of normal stem cell lines from developmentally abnormal embryos ordinarity discarded opens a new source of embryos for stem cells, whether for research or for eventual therapeutic use within the donating families.     

Changes in biomechanical parameters during heart perfusion and after midazolam pre-medication--experimental pilot study

Background: Midazolam is a frequently used benzodiazepine in anaesthesiology and intensive care. Aim: The aim of pilot study was to monitor its effect during heart perfusion in the laboratory rat. Methods: The same groups of animals (n = 10). The 1(st) group was treated with midazolam in a dose of 0.5mg/kg i.p. The 2(nd) group was a placebo. After i.p. administration of heparine injection of 500 IU dose, the hearts were excised and perfused (modified Langendorf's method). Working schedule: stabilization/ischaemia/reperfusion proceed at intervals of 20/30/60 min. Monitored parameters in isolated heart: left ventricle pressure (LVP), end-diastolic pressure (LVEDP), contractility (+dP/dt(max)). Results: The treated hearts showed improved postischemic recovery, reaching LVP values of 92 +/- 6 % at the end of the reperfusion, placebo only 61 +/- 7 %. In placebo hearts LVEDP rose from 10.0 +/- 0.5 mmHg to 43 +/- 4 mmHg after, in treated animals only about 25 mmHg. The treated hearts improved +dP/dt(max) recovery during reperfusion to 91 +/- 8 %. These values were significantly greater than those obtained from the placebo hearts. Conclusions: Positive changes in monitored parameters were found in this experimental pilot study. We conclude that the administration of midazolam in laboratory rats has a cardioprotective potential against ischemia-reperfusion induced injury.     

The correlation of serum immunoglobulin free light chain levels and selected biological markers in multiple myeloma

Aims: the presented study is aimed at the evaluation of correlation of free light chains serum levels - kappa, lambda and their relation (K/L ratio) and serum levels of selected biological markers in a group of patients with multiple myeloma examined at the time of the diagnosis. Methods: 102 patients with multiple myeloma were included in this prospective study. Free light chains serum levels were determined by Freelite Binding Site system, for determination of serum levels of selected parameters the following methods were used: REA thymidinekinase (TK), RIA (beta(2)microglobulin (beta(2)m), ICTP, PINP), enzymoimmunoassay (sIL-6R, sVCAM-1, sICAM-1, sOPG) and quantitative enzymatic immunoassay (sHGF, sVEGF, sSyndecan-1 (sSyn-1) a sFas). Results: There was found a correlation in the kappa-group of the dominant kappa chain and serum readings of beta(2)m (r = 0.344, p = 0.005), TK (r = 0.263, p = 0.035), ICTP (r = 0.402, p = 0.001), PINP (r = 0.264, p = 0.039), sOPG (r = 0.328, p = 0.028), sSyn-1 (r = 0.255, p = 0.046) and sFas (r = 0.418, p = 0.001). In case of K/L ratio there was found a statistically significant association of levels of beta(2)m (r = 0.316, p = 0.01), TK (r = 0.274, p = 0.027), ICTP (r = 0.346, p = 0.006), PINP (r = 0.261, p = 0.042), sSyn-1 (r = 0.283, p = 0.026) and sFas (r = 0.377, p = 0.002). In the lambda-group the analysis confirmed mutual dependence of the dominant lambda chain levels on beta(2)m (r = 0.476, p = 0.003), ICTP (r = 0.375, p = 0.022), sVCAM-1 (r = 0.383, p = 0.019), sHGF (r = 0.441, p = 0.006) and sFas (r = 0.334, p = 0.040). In addition we ascertained a correlation of L/K ratio and levels of beta(2)m (r = -0.473, p = 0.003), TK (r = -0.412, p = 0.011), ICTP (r = -0.331, p = 0.045), PINP (r = -0.409, p = 0.012), sHGF (r = -0.357, p = 0.028), sSyn-1 (r = -0.449, p = 0.005) a sFas (r = - 0.371, p = 0.022). Conclusions: The study confirmed mutual correlation of FLC serum levels and the levels of several selected biological markers, in particular beta(2)m, TK, ICTP, PINP, sSyn-1 a sFas at time of the diagnosis. It referred to the mutual relation of bone marrow microenvironment, biological qualities of clonal plasmocytes and the intensity of the free light chains production by the tumour cell population.     

Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein

The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.     

Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate

The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.     

On-line monitoring of microbial volatile metabolites by proton transfer reaction-mass spectrometry

A method for analysis of volatile organic compounds (VOCs) from microbial cultures was established using proton transfer reaction-mass spectrometry (PTR-MS). A newly developed sampling system was coupled to a PTR-MS instrument to allow on-line monitoring of VOCs in the dynamic headspaces of microbial cultures. The novel PTR-MS method was evaluated for four reference organisms: Escherichia coli, Shigella flexneri, Salmonella enterica, and Candida tropicalis. Headspace VOCs in sampling bottles containing actively growing cultures and uninoculated culture medium controls were sequentially analyzed by PTR-MS. Characteristic marker ions were found for certain microbial cultures: C. tropicalis could be identified by several unique markers compared with the other three organisms, and E. coli and S. enterica were distinguishable from each other and from S. flexneri by specific marker ions, demonstrating the potential of this method to differentiate between even closely related microorganisms. Although the temporal profiles of some VOCs were similar to the growth dynamics of the microbial cultures, most VOCs showed a different temporal profile, characterized by constant or decreasing VOC levels or by single or multiple peaks over 24 h of incubation. These findings strongly indicate that the temporal evolution of VOC emissions during growth must be considered if characterization or differentiation based on microbial VOC emissions is attempted. Our study may help to establish the analysis of VOCs by on-line PTR-MS as a routine method in microbiology and as a tool for monitoring environmental and biotechnological processes.     

Life form-specific variations in leaf water oxygen-18 enrichment in Amazonian vegetation

Leaf water (18)O enrichment (Delta(o)) influences the isotopic composition of both gas exchange and organic matter, with Delta(o) values responding to changes in atmospheric parameters. In order to examine possible influences of plant parameters on Delta(o) dynamics, we measured oxygen isotope ratios (delta(18)O) of leaf and stem water on plant species representing different life forms in Amazonia forest and pasture ecosystems. We conducted two field experiments: one in March (wet season) and another in September (dry season) 2004. In each experiment, leaf and stem samples were collected at 2-h intervals at night and hourly during the day for 50 h from eight species including upper-canopy forest trees, upper-canopy forest lianas, and lower-canopy forest trees, a C(4) pasture grass and a C(3) pasture shrub. Significant life form-related differences were detected in (18)O leaf water values. Initial modeling efforts to explain these observations over-predicted nighttime Delta(o) values by as much as 10 per thousand. Across all species, errors associated with measured values of the delta(18)O of atmospheric water vapor (delta(v)) appeared to be largely responsible for the over-predictions of nighttime Delta(o) observations. We could not eliminate collection or storage of water vapor samples as a possible error and therefore developed an alternative, plant-based method for estimating the daily average delta(v) value in the absence of direct (reliable) measurements. This approach differs from the common assumption that isotopic equilibrium exists between water vapor and precipitation water, by including transpiration-based contributions from local vegetation through (18)O measurements of bulk leaf water. Inclusion of both modified delta(v) and non-steady state features resulted in model predictions that more reliably predicted both the magnitude and temporal patterns observed in the data. The influence of life form-specific patterns of Delta(o) was incorporated through changes in the effective path length, an important but little known parameter associated with the Péclet effect.     

Specialization pays off: contrasting effects of two types of tannins on oak specialist and generalist moth species

Phytochemical coevolution theory posits that specialist herbivores will be less sensitive than generalists to the defensive compounds of their host. On the other hand, both types of herbivores should allegedly be similarly sensitive to ‘quantitative’ defences, such as tannin compounds. In this paper, we critically examine the biological effects of two types of tannins: vescalagin (a quantitatively dominant hydrolysable tannin of Quercus robur), and a mix of condensed tannins. In a phylogenetically controlled design, we compare the response of two specialist moth species (Dichonia aprilina and Catocala sponsa) and two generalist species (Acronicta psi and Amphipyra pyramidea) to four artificial diets: a control diet, a diet with 50 mg g^?1 vescalagin, a diet with 15 mg/g condensed tannins, and a diet with both 50 mg g^?1 vescalagin and 15 mg g^?1 condensed tannins. Overall, we find drastic effects of vescalagin and pronounced differences in the responses of generalist and specialist herbivores, but no detectable effects of condensed tannins, and no interaction between the two types of compounds. More specifically, vescalagin reduced the growth of generalist species to one‐half of control levels over 72 h. The compound served as a strong feeding deterrent to generalists, reducing ingestion rates by two‐thirds. Vescalagin also reduced the metabolic and growth efficiency of generalist species to between 16% and 56% of control levels – effects which were lacking or even reversed in specialist species. These patterns suggest that vescalagin forms an important part of the oak's defence against herbivores, and that specialist species have adapted to deal with such substances. In terms of biological effects, condensed tannins seem much less important. Given a quantitative dominance of hydrolysable tannins over condensed tannins in oak leaves, and a seasonal decline in overall tannin levels, these findings contradict the previous notion that widespread spring feeding among oak herbivores could be attributed to tannins.