Effects of high arsenic and fluoride soil concentrations on soybean plants

Arsenic (As) and Fluoride (F) are present in many soils, affecting crops and posing risks in the food chain. We performed pot experiments on spiked soils enriched in these elements either individually or simultaneously, over a wide range of concentrations. Soybean biomass production, grain yield, As and F accumulation and distribution within the plant, and the antioxidant response to these stresses were analyzed. Arsenic was more toxic than F. At As levels >35 mg/kg and F levels >375 mg/kg, yield loss reached 60% and 30%, respectively. At the highest dose of As plants died within 2 weeks, whereas F showed no lethality. When they were applied simultaneously, detrimental effects were more important. As and F in plants increased in all soybean organs although grains presented the lowest concentrations. Antioxidant enzymes were enhanced in plants but this increase was not high enough to cope with the oxidative damage.     

Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.     

The performance of fungal xylan-degrading enzyme preparations in elemental chlorine-free bleaching for Eucalyptus pulp

Cellulase-free xylan-degrading enzyme preparations from Acrophialophora nainiana, Humicola grisea var. thermoidea and two Trichoderma harzianum strains were used as bleaching agents for Eucalyptus kraft pulp, prior to a chlorine dioxide and alkaline bleaching sequence. In comparison to the control sequence (performed without xylanase pretreatment), the sequence incorporating enzyme treatment was more effective. Removal of residual lignin was indicated by a reduction in kappa number. Overall, enzyme preparations from T. harzianum were marginally more effective in reducing pulp viscosity and chlorine chemical consumption and improving the brightness of the kraft pulp. However, the highest reduction in pulp viscosity was mediated by the xylanase preparation from A. nainiana. Xylanase pretreatment compares very favorably with that of chemical pulping. Journal of Industrial Microbiology & Biotechnology (2002) 28, 204–206 DOI: 10.1038/sj/jim/7000227 Received 27 April 2001/ Accepted in revised form 03 November 2001     

Synchronization of human diploid fibroblasts at multiple stages of the cell cycle

Because of the scarcity of techniques for synchronizing the growth of cultured human diploid fibroblasts at multiple stages within the cell cycle, efforts were expended in this report to establish a set of protocols that would permit synchronization of cells at several different points throughout the cycle. The protocols that were developed to synchronize the growth of HSF-24 and HSF-55 cells, human foreskin-derived fibroblast cultures, were modifications of procedures employed to synchronize the growth of cultured rodent cells. Optimization of synchrony induction was directed by consideration of both the biochemical properties of the synchronized populations (determined via three-parameter flow cytometric measurements of DNA, RNA, and protein contents) and their kinetic behavior following reversal of the synchronization-inducing blockade (determined via combined flow cytometric analysis of DNA content, [3H]thymidine autoradiography, and measurement of increase in cell number). The conditions judged to yield the best results for studying events associated with production of a G0 block or for maintaining cells for prolonged periods in G0 were those in which the cells were grown to confluency in D-MEM supplemented with 10% fetal bovine serum. Procedures producing the best results for studying processes associated with the G0 to G1 transition, G1 events, and operations accompanying the transition from G1 to S, employed subconfluent growth for 48 h in alpha-MEM + 0.1% fetal bovine serum (alpha-MEM0.1F) followed by resuspension in alpha-MEM containing 10% fetal bovine serum (alpha-MEM10F). When the goal was to obtain cells in which to study very early S-phase events, satisfactory results were achieved by combining a 48-h period of subconfluent growth in alpha-MEM0.1F, followed by treatment for 24 h in alpha-MEM10F containing 5 micrograms/ml aphidicolin. For study of events occurring in mid- to late-cycle, acceptable results were achieved by combining a 48-h block in alpha-MEM0.1F with resuspension for 24 h in alpha-MEM10F containing 10(-3) M hydroxyurea followed by resuspension in drug-free alpha-MEM10F. The best results were obtained with these latter synchronization procedures (i.e., low-serum/high-serum + APC or HU/high serum) when the fetal calf serum was replaced with heat-inactivated calf serum. The success achieved in synchronizing the growth of these human diploid fibroblasts compared favorably/exceeded the results obtained with synchronized cultures of Chinese hamster ovary cells.     

Selection and characterization of a varient of murine L5178Y lymphoma resistant to local anesthetics.

A varient of murine L5178Y lymphoma resistant to procaine hydrochloride (PH) was selected by exposing the cells to gradual increments of PH in the growth medium until the cell grew exponentially in the presence of 1.5 mM PH. Using cinephotomicrography, it was observed that the majority of cells that initially succumbed to PH failed to undergo successful mitosis. With respect ot chromosomal, cell size distribution and flow microfluorometric analyses, the PH-resistant cells are very similar to a spontaneous tetraploid cell line (R1T) previously cloned. The isolated cells, designated R1/P, were also found to be cross-resistant to analogues of PH, namely, lidocaine, tetracaine and dibucaine. The naturally-occurring tetraploid cell line (R1T) was also found to be more resistant to local anesthetics, although not to the same extent as R1/P cells. Since the enzyme that hydrolyzes procaine appears to be absent in all these lymphoid cell lines, the difference in resistance does not appear to depend on differences in the ability of these cells to remove the agent. It is suggested that an alteration in the structure and/or function of the plasma membrane in R1/P cells have rendered them either less sensitive to the membrane-perturbing effects of the local anesthetics or less permeable to local anesthetics molecules. The ability of local anesthetics to affect membranes and cytoskeleton structures may play a role in the genesis and/or selection of these cell variants.     

Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated

Over the past decade, there have been many reports suggesting the presence of complex carbohydrates on nuclear and cytoplasmic proteins in mammalian cells. Some of the most often cited of these reports deal with the glycosylation of the high mobility group (HMG) proteins. These are relatively abundant chromosomal proteins that are known to be associated with nucleosomes and actively transcribed regions of chromatin. The original report describing HMG protein glycosylation presented several lines of evidence suggesting that these proteins are glycosylated, including carbohydrate compositional analysis and periodic-acid Schiff staining. We have attempted to repeat these observations with more highly purified protein than was utilized in the original study. Using carbohydrate compositional analysis performed by high pH anion exchange chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins. In addition, we found no evidence for the presence of O- GlcNAc, a well known form of nuclear glycosylation. The HMG proteins did react with periodate, suggesting the presence of a modification containing cis-diols on the protein. Several tryptic peptides isolated from HMG 14 and 17 which retained the periodate reactivity had in common lysine residues, suggesting a potential modification of the straightepsilon-amino groups of lysines such as nonenzymatic glycation. Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.      

Anticancer effects and lysosomal acidification in A549 cells by Astaxanthin from Haematococcus lacustris

Astaxanthin (AXN) is known to have health benefits by epidemiological studies. Therefore, it is of interest to assess the effect of AXN (derived from indigenous unicellular green alga Haematococcus lacustris) to modulate cell cycle arrest, lysosomal acidification and eventually apoptosis using in vitro in A549 lung cancer cells. Natural extracts of astaxanthin were obtained by standardized methods as reported earlier and characterized by standard HPLC and MS. Treatment of A549 cells with AXN (purified fraction) showed significant reduction in cell viability (about 50%) as compared to crude extract at 50µM concentration. Thus, we show the anticancer effects and lysosomal acidification in A549 cells by Astaxanthin from Haematococcus lacustris for further consideration. Together, our results demonstrated the anticancer potential of AXN from Haematococcus lacustris, which is found to be mediated via its ability to induce cell cycle arrest, lysosomal acidification and apoptotic induction.