Specific Y chromoome fluorescence in interphase nuclei of epithelial cells in human buccal smears

Summary A simplified method is described to demonstrate Y chromosome fluorescence in interphase nuclei of epithelial cells in human buccal smears using Quinacrine Mustard. This technique turned out to be efficient in our hands and would be of value in determining the sex chromosomes in the epithelial cells of the skin in cases of allogeneic transplantations from females to males and vice versa to determine the period during which the grafts survived.     

Telomere maintenance,function and evolution: the yeast paradigm

Telomeres are multifunctional genetic elements that cap chromosome ends, playing essential roles in genome stability, chromosome higher-order organization and proliferation control. The telomere field has largely benefited from the study of unicellular eukaryotic organisms such as yeasts. Easy cultivation in laboratory conditions and powerful genetics have placed mainly Saccharomyces cerevisiae, Kluveromyces lactis and Schizosaccharomyces pombe as crucial model organisms for telomere biology research. Studies in these species have made it possible to elucidate the basic mechanisms of telomere maintenance, function and evolution. Moreover, comparative genomic analyses show that telomeres have evolved rapidly among yeast species and functional plasticity emerges as one of the driving forces of this evolution. This provides a precious opportunity to further our understanding of telomere biology.     

Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains approximately 2.5-fold higher density of background DNA breaks with 3'-OH ends, and also approximately 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei.     

Defective glucocorticoid receptor nuclear translocation and altered histone acetylation patterns in glucocorticoid-resistant patients

BACKGROUND: Most chronic inflammatory diseases are well controlled by glucocorticoids. However, a minority of patients fails to respond adequately to this treatment. OBJECTIVE: We wished to determine whether glucocorticoid insensitivity in a group of steroid-resistant (SR) and steroid-dependent (SD) asthmatic subjects resulted from an inability of the glucocorticoid receptor (GR) to translocate into the nucleus. METHODS: Glucocorticoid receptor nuclear translocation was determined in PBMCs by immunocytochemistry and GR function measured by suppression of TNF-alpha-induced GM-CSF release and effects of dexamethasone on histone acetylation. RESULTS: Glucocorticoid repression of TNF-alpha-induced GM-CSF release was reduced in PBMCs from SD and SR patients. This inhibition correlated with a failure of GR to translocate into the nucleus and induce histone acetylation in response to dexamethasone. In addition, dexamethasone failed to inhibit TNF-alpha-induced histone acetyltransferase activity, which predominantly targeted histone residues lysine (K)8 and K12. However, in a subset of patients, even high levels of GR nuclear translocation failed to produce histone acetylation in response to dexamethasone. Histone H4 K5 acetylation, a marker of dexamethasone transactivation, was specifically reduced in this group. However, cells from this subset of steroid-insensitive subjects were still capable of inhibiting TNF-alpha-induced histone acetylation. CONCLUSION: We have identified a novel mechanism of glucocorticoid insensitivity in a group of SR and SD subjects. These data suggest that most patients respond to glucocorticoids according to the degree of GR nuclear translocation occurring, but some subjects with steroid resistance have a reduced response because of a failure of steroids to transactivate, rather than transrepress.     

Linking the epigenetic 'language' of covalent histone modifications to cancer

Covalent modifications of histones, such as acetylation, methylation, and phosphorylation, and other epigenetic modulations of the chromatin, such as methylation of DNA and ATP-dependent chromatin reorganisation, can play a major part in the multistep process of carcinogenesis, with far-reaching implications for human biology and human health. This review focuses on how aberrant covalent histone modifications may contribute to the development of a variety of human cancers, and discusses the recent findings with regard to potential therapies.     

Distinct microtubule and chromatin characteristics of human oocytes after failed in-vivo and in-vitro meiotic maturation

BACKGROUND: While a complete failure of meiotic maturation following hCG administration is rare during IVF cycles, cases arise in which patients repeatedly display a high incidence of failure to complete maturation to metaphase II (MII) in vivo. For the immature oocytes of such patients, our objectives were (i) to ask whether progression to MII could be supported in vitro, and (ii) to define their microtubule/chromatin properties following in-vitro maturation (IVM). Together, these studies were aimed at augmenting our understanding of factors underlying meiotic arrest in the human. METHODS: Cases are presented here for two patients (A and B) producing oocytes that recurrently showed the inability to mature to metaphase II in vivo. Following IVM attempts, chromatin and microtubule characteristics were identified in those oocytes that remained arrested during meiosis I. RESULTS: In patient A, meiotically arrested oocytes exhibited clear defects in spindle and chromatin arrangements. In contrast, the majority of oocytes from patient B displayed normal MI and MII spindles with aligned chromosomes, although some oocytes exhibited indications for possible defects in cell cycle control. CONCLUSIONS: Together, these analyses illustrate two cases with oocytes exhibiting a common gross defect, that is meiotic maturation arrest, but revealing different aetiologies or manifestations as evidenced by the presence or absence of abnormal spindle/chromatin organization. This work reinforces the existence of intrinsic defects in oocytes of some patients, the molecular and cellular bases of which merit further investigation.