Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis

Interactions between nuclear proteins and chromatin frequently occur on the time scale of seconds and below. These transient binding events are important for the fast identification of target sites as concluded from our previous analysis of the human chromatin remodelers Snf2H and Snf2L from the imitation switch (ISWI) family. Both ATP-driven molecular motor proteins are able to translocate nucleosomes along the DNA and appear to exert this activity only on a small number of nucleosomes to which they bind more tightly. For mechanistic studies, one needs to distinguish such translocation reactions or other long-lived interactions associated with conformational changes and/or ATP hydrolysis from nonproductive chromatin sampling during target search. These processes can be separated by measuring the duration of nucleosome binding with subsecond time resolution. To reach this goal, we have developed a fluorescence bleaching technique termed pixel-wise photobleaching profile evolution analysis (3PEA). It exploits the inherent time structure of confocal microscopy images and yields millisecond resolution. 3PEA represents a generally applicable approach to quantitate transient chromatin interactions in the 2- to 500-ms time regime within only ∼1 s needed for a measurement. The green autofluorescent protein (GFP)-tagged Snf2H and Snf2L and the inactive Snf2L+13 splice variant were studied by 3PEA in comparison to the isolated GFP or red autofluorescent protein and a GFP pentamer. Our results reveal that the residence time for transient chromatin binding of Snf2H and Snf2L is <2 ms, and strongly support the view that ISWI-type remodelers are only rarely active in unperturbed cells during G1 phase.     

Assessment of sperm functions in infertile patients with varicoceles

Acrosin activity, acrosome reaction and nuclear chromatin condensation were studied in 24 infertile patients with varicoceles and 26 fertile men with or without varicocele. Chromatin condensation, assessed by aniline blue staining, and acrosin activity, evaluated by gelatinolysis technique, were significantly affected in the group of infertile patients. Defective chromatin condensation and defective acrosin activity were detected in 67% and 50% of the infertile patients, respectively. No significant difference was found between the two groups regarding the acrosome reaction, which was assessed by the triple staining technique after exposure of spermatozoa to low temperature (4 degrees C). This study identified a subgroup of infertile patients with normal standard semen parameters but impaired sperm functions. Results of the sperm function tests and standard semen parameters were not correlated. Therefore, it is concluded from this study that important sperm functions are impaired in patients with varicocele and that the gelatinolysis technique and aniline blue staining are effective tools for assessment of the fertilization potential of varicocele patients.     

The origin of DNA replication and Fieller's problem

The paper considers the statistical problem of estimating the origin of DNA replication within the human ribosomal DNA (rDNA) locus and the issue of assessing the standard error of the estimate. Based on mapping the cumulative replication index (CRI), two different modelling schemes are suggested and investigated. The statistical problem of constructing a confidence interval for the origin of DNA replication is related to Fieller's problem of obtaining a confidence interval for the ratio of two normal means. Standard normal theory, the delta and bootstrap methods are used to estimate the standard error of the estimate of the origin of DNA replication, as well as the variation of the replication rate.     

Aberrant methylation and histone deacetylation of cyclooxygenase 2 in gastric cancer.

Cyclooxygenase 2 plays a critical role in the development of gastrointestinal cancers in both human and animal models. About 80% of the gastric cancer showed a high level of expression of cyclooxygenase 2, but a subset of cases do not express without unknown reason. Aberrant methylation of CpG island of COX-2 was examined by using a series of gastric cancer cell lines and primary gastric cancers. Two out of 8 cell lines (25%) and 11 out of 93 (12%) primary cancers showed aberrant methylation of the 5' region of COX-2. Methylation of COX-2 was closely associated with loss of expression and treatment of methylation inhibitor, 5-deoxy-2'-azacytidine restored the expression of COX-2. A combined treatment of 5-deoxy-2'-azacytidine and a histone deacetylese inhibitor, trichostatin A, restored re-expression of the gene synergistically and chromatin immunoprecipitation analysis revealed that histone of methylated COX-2 promoter is deacetylated, indicating the role of cytosine methylation and histone deacetylation in the silencing of the gene. These results indicate that a subset of gastric cancer with COX-2 methylation evolves through the pathway that is independent of COX-2 expression and that COX-2 inhibitor may not be useful to induce apoptosis in these cases.     

Frequency and Patterns of Premature Sperm Chromosome Condensation in Oocytes Failing to Fertilize After Intracytoplasmic Sperm Injection

Purpose: The purpose of this study was to evaluate reasonsfor fertilization failure after intracytoplasmic sperminjection as a part of internal quality control and to reviewcorresponding previous data. Methods: One hundred injected but unfertilized oocytes werefixed and examined after Giemsa staining. Results: Three oocytes (3.0%) did not show the presenceof a spermatozoon and two (2.0%) contained pronuclearstructures. An intact spermatozoon was found in 25 cases(25.0%), whereas the sperm nucleus had undergonepremature chromosome condensation (PCC) in 70 cells (70.0%).A modified classification system was established tocharacterize the different PCC patterns. Conclusions: PCC indicates a correct intracytoplasmicinjection and excludes technical problems as a major reasonfor fertilization failure in the present study. A lack of oocyteactivation due to cytoplasmic immaturity is consideredresponsible for the occurrence of PCC. A review of theliterature shows that the role of sperm chromatinabnormalities in the process of nuclear decondensation needs furtherinvestigation.     

Chromatin Packaging as an Indicator of Human Sperm Dysfunction

Purpose: Understanding the causes of fertilization failure is an important research field in assisted reproductive programs. The present study aimed to evaluated the possible relationship between chromatin packaging quality (CMA₃ staining) and (i) normal morphology and (ii) its ability to predict the functional integrity of spermatozoa in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment programs. Methods: Semen of 140 men from IVF and ICSI couples were analyzed for sperm concentration, motility, morphology, and chromatin packaging (CMA₃). For CMA₃ classification, two cutoff values were used, namely, 44.5%±13 and 1 SD above the mean, i.e., 57.5% (rounded off to 60%). IVF and ICSI data were stratified using three basic cutoff values for CMA₃ staining, namely, <44%, >44–60%, and >60%. Results: Based on CMA₃ results patients were divided into four groups, namely, group A, <44% CMA₃ (n = 15, IVF); group B, 44% and <60% CMA₃ (n = 39, IVF); group C, 60% CMA₃ (n = 45 IVF); and group D, 60% CMA₃(n = 41 ICSI). During receiver operator characteristic analyses the estimated cutoff value for CMA₃ staining, to distinguish between <4% and 4% morphology groups, was 60%. The area under the curve was 0.89, sensitivity of 75%, and specificity of 100%. When IVF rates of >60% and <60% were used, the optimal CMA₃ value for prediction of fertilization success again was recorded at 60%. The area under the curve was 0.76, sensitivity of 81.5%, and specificity of 63.6%. Conclusions: Chromatin packaging assessments should be included as a complementary assay to the sequential diagnostic approach of the male-factor patients.