Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8 M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. deltasod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.     

Homozygote for mutation c.1204 + 1G > A of the ARSA gene presents with a late-infantile form of metachromatic leukodystrophy and a rare MRI white matter lesion type

The metachromatic leukodystrophy (MLD)--causing mutation c.1204 + 1G > A damages an intron-exon splice site recognition sequence. This results in a complete loss of enzymatic activity of arylsulfatase A (ARSA) protein molecules. We have found a late-infantile type MLD-patient to be homozygous for this mutation, which was not reported earlier, but is consistent with previous suggestions. Interestingly, the cerebral magnetic resonance imaging (MRI) in this patient displayed linear or punctuate structures radiating in the demyelinated white matter, which resembled the patterns described in Pelizaeus-Merzbacher disease. It should be emphasised that whenever a cerebral MRI demonstrates the "tigroid" or "leopard-skin" demyelination pattern not only Pelizaeus-Merzbacher disease, but also metachromatic leukodystrophy diagnosis should be considered; this suggests the necessity of ARSA activity estimations in patients with such specific MRI patterns.     

Non-homologous DNA end joining repair in normal and leukemic cells depends on the substrate ends

Double-strand breaks (DSBs) are the most serious DNA damage which, if unrepaired or misrepaired, may lead to cell death, genomic instability or cancer transformation. In human cells they can be repaired mainly by non-homologous DNA end joining (NHEJ). The efficacy of NHEJ pathway was examined in normal human lymphocytes and K562 myeloid leukemic cells expressing the BCR/ABL oncogenic tyrosine kinase activity and lacking p53 tumor suppressor protein. In our studies we employed a simple and rapid in vitro DSB end joining assay based on fluorescent detection of repair products. Normal and cancer cells were able to repair DNA damage caused by restriction endonucleases, but the efficiency of the end joining was dependent on the type of cells and the structure of DNA ends. K562 cells displayed decreased NHEJ activity in comparison to normal cells for 5' complementary DNA overhang. For blunt-ended DNA there was no significant difference in end joining activity. Both kinds of cells were found about 10-fold more efficient for joining DNA substrates with compatible 5' overhangs than those with blunt ends. Our recent findings have shown that stimulation of DNA repair could be involved in the drug resistance of BCR/ABL-positive cells in anticancer therapy. For the first time the role of STI571 was investigated, a specific inhibitor of BCR/ABL oncogenic protein approved for leukemia treatment in the NHEJ pathway. Surprisingly, STI571 did not change the response of BCR/ABL-positive K562 cells in terms of NHEJ for both complementary and blunt ends. Our results suggest that the various responses of the cells to DNA damage via NHEJ can be correlated with the differences in the genetic constitution of human normal and cancer cells. However, the role of NHEJ in anticancer drug resistance in BCR/ABL-positive cells is questionable.     

Age, insulin, SHBG and sex steroids exert secondary influence on plasma leptin level in women

AIM: As the link between body fat and leptin is well known, the aim of the study was to seek for secondary regulators of plasma leptin level. PATIENTS: 86 women (mean: age 47.0+/-14.3 years; estradiol 50.0+/-60.6 ng/l; FSH 52.4+/-42.9 IU/l; BMI 26.9+/-5.9) divided into three groups according to their BMI. Group A: 39 normal weight women (mean: age 44.4+/-16.0 years; estradiol 69.6+/-79.8 ng/l; FSH 50.4+/-47.7 IU/l; BMI 22.9+/-1.3). Group B: 27 overweighted women (mean: age 55.0+/-6.4 years; estradiol 25.1+/-17.2 ng/l; FSH 75.6+/-26.3 IU/l; BMI 27.7+/-1.6). Group C: 21 obese women with mean: age 48.7+/-12.2 years; estradiol 36.9+/-44.0 ng/l; FSH 42.3+/-36.6 IU/l and BMI 34.6+/-4.9. METHODS: Standard clinical evaluation and hormone evaluation (LH, FSH, prolactin, estradiol, leptin, insulin-like growth factor-I (IGF-I), human growth hormone (hGH), insulin-like growth factor binding protein-3 (IGFBP-3), insulin, dihydroepiandrosterone sulphate (DHEAS), sex hormone binding globin (SHBG) and testosterone were done in basic condition which levels of were measured by RIA kits. Statistical analysis. Shapiro-Wilk test, Mann-Whitney-Wilcoxon u test, Spearman rank correlation coefficient and stepwise multiple regression: p values of 0.05 or less were considered as significant. RESULTS: Taking all women into account (n=86) the plasma leptin level correlated directly with age (r=0.32; p<0.02), body mass (r=0.60; p<0.001), BMI (r=0.71; p<0.001) as well as inversely with estradiol (r=-0.21; p<0.05), IGF-I (r=-0.24; p<0.05), SHBG (r=-0.34; p<0.01) and DHEAS (r=-0.30; p<0.01). However only in the group B leptin/age relation remained (r=0.40; p<0.05) after the division according to BMI. In the group B the leptin /DHEAS (r=-0.40; p<0.05) and leptin/PRL (r=0.51; p<0.05) links were also present. In the group C the leptin/SHGB relation (r=-0.56; p<0.02) only remained and an association between insulin and leptin was found (r=0.48; p<0.05). The body mass and BMI relation to age were again present only in all 86 women (r=0.30; p<0.002: r=0.36; p<0.001 resp.). Having split the women into groups, these links either disappeared or became inverse (rC=-0.39; p<0.05). Taking into consideration age/leptin relation in all women, the division according to the menopausal status revealed the direct relation in premenopausal women (n=29; r=0.43; p<0.02) and a reverse one in postmenopausal women (n=38; r=-0.32; p<0.05). The plasma leptin level was the highest (p<0.001) in group C (23.2+/-10.4 microg/l) and the lowest was found in the group A (8.9+/-4.1 microg/l). That corresponded with the differences in mean body mass index and mean body mass. The stepwise multiple regression revealed that body mass index accounted for 31% (p<0.001) and plasma SHBG level accounted for 17.7% (p<0.02) of plasma leptin variance in all women. In the group A body mass and age together accounted for 61% (p<0.01) and estradiol alone accounted for 44% (p<0.02) of plasma leptin variance. In the group B insulin alone accounted for 39% (p<0.05) and together with testosterone accounted for 46% (p<0.05) of plasma leptin variance. Finally in obese women none of the evaluated parameters significantly accounted for leptin variance. CONCLUSION: The results presented in this paper confirmed the strong influence of body fat mass on serum leptin concentration. However insulin, SHBG, sex steroids as well as age may also exert secondary influence on plasma leptin level in certain groups of women.     

Potential application of stem cells in urogynecology

Recent advances in stem cells therapy and tissue engineering techniques hold great promise for recovery of external urethral sphincter proper functioning and urinary incontinent patients treatment. Adult stem cells (ASCs) may be derived from striated muscles, fat tissue or as mesenchymal cells from bone marrow. These cells can differentiate in functionally normal smooth or striated muscle cells. ASCs injected to external urethral sphincter or bladder neck cause the increase in urethral closure pressure. Favourable findings of trials in animal models and in vitro encouraged to first trials in humans and hold a promising future for the treatment of urinary incontinence.     

Magnetic field inhibits isolated lymphocytes' proliferative response to mitogen stimulation

We aimed to find out how the exposure of isolated lymphocytes to a pulsed magnetic field (MF) affected their in vitro proliferative response to mitogenic stimulation. Cells were exposed to MF of various intensities (0.3, 0.6, and 1.2 T) at a constant frequency of 30 Hz, for a period of 60, 180, and 330 s. Then, the proliferative response of splenocytes was induced by optimal concentrations of concanavalin A (Con A; mitogenic toward T cells), bacterial lipopolysaccharide (LPS; mitogenic toward B cells), or pokeweed mitogen (PWM; mitogenic toward both populations). We found that the exposure of lymphocytes to the MF profoundly inhibited their proliferative response to mitogens. The suppressive action of the MF on B and T cell proliferation was intensified when a cooperative response of those two lymphocyte populations was simultaneously induced by PWM. The inhibitory effect of MF depended on the exposure time and MF intensity. Prolonged exposure and/or a stronger intensity of the MF weakened its inhibitory influence on the response of lymphocyte to mitogenic stimulation. The data show that an exposure to MF may influence the activity of lymphocytes in their response to mitogenic stimuli.     

Nonhematopoietic stem cells of fetal origin--how much of today's enthusiasm will pass the time test?

Stem cells originating at fetal age are for many reasons superior as a material for the regenerative medicine purposes, when compared to their adult counterparts. While hematopoietic cells, isolated from fetal liver or cord blood, have been well known for a long time and have passed practical tests as clinical transplantation material, the non-hematopoietic cells are newly recognized, and the knowledge of their phenotype and differentiation potential is rather insufficient. We, and the others, have identified a subpopulation of cord blood cells phenotypically different from hematopoietic cells (CD34-, CD45-, CD29+, CD44+, CD51+, CD105+, SH-2, SH-3), in vitro plastic adherent, and capable of multilineage differentiation. The other candidates for multipotential stem cells are cells extracted from umbilical cord or placental tissue. The preliminary observations suggest, that these cells, phenotypically similar to the nonhematopoietic cord blood cells, are capable of extensive replication in vitro and of multilineage differentiation into a variety of tissues including cardiac muscle, bone and cartilage, adipocytes, and nerve cells. The other possible medical applications include "rejuvenation" of selected tissues and systems in senile patients, and therapeutical cloning - for both purposes, cells at the fetal stage of genetic regulation may be more useful than cells collected from adult donors. There is still, however, a high level of uncertainty concerning future medical applications of fetal stem cells. Their numbers and characteristics may differ from the preliminary observations, and their behavior in vivo may not fulfill the expectations originating from the in vitro studies. Finally, the autologous applications of stem cells collected at the stage of birth may need the involvement of technical and financial resources for the storage of frozen cell samples throughout the period of life of their potential user. Such procedure seems possible from technical point of view, but may be inadequately substantiated by the eventual advantages.     

Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells

The heterodimer consisting of ecdysteroid receptor (EcR) and ultraspiracle (USP), both of which are members of the nuclear receptor superfamily, is considered to be the functional ecdysteroid receptor. Here we analyzed the subcellular distribution of EcR and USP fused to fluorescent proteins. The experiments were carried out in mammalian COS-7, CHO-K1 and HeLa cells to facilitate investigation of the subcellular trafficking of EcR and USP in the absence of endogenous expression of these two receptors. The distribution of USP tagged with a yellow fluorescent protein (YFP-USP) was almost exclusively nuclear in all cell types analyzed. The nuclear localization remained constant for at least 1 day after the first visible signs of expression. In contrast, the intracellular distribution of EcR tagged with a yellow fluorescent protein (YFP-EcR) varied and was dependent on time and cell type, although YFP-EcR alone was also able to partially translocate into the nuclear compartment. Coexpression of YFP-EcR with USP tagged with a cyan fluorescent protein (CFP-USP) resulted in exclusively nuclear localization of both proteins in all cell types analyzed. The USP-induced nuclear localization of YFP-EcR was stable for at least 20 hours. These experiments suggest that USP has a profound effect on the subcellular distribution of EcR.