The end adjusts the means: Heterochromatin remodelling during terminal cell differentiation

All cells that constitute mature tissues in an eukaryotic organism undergo a multistep process of cell differentiation. At the terminal stage of this process, cells either cease to proliferate forever or rest for a very long period of time. During terminal differentiation, most of the genes that are required for cell ‘housekeeping’ functions, such as proto-oncogenes and other cell-cycle and cell proliferation genes, become stably repressed. At the same time, nuclear chromatin undergoes dramatic morphological and structural changes at the higher-order levels of chromatin organization. These changes involve both constitutively inactive chromosomal regions (constitutive heterochromatin) and the formerly active genes that become silenced and structurally modified to form facultative heterochromatin. Here we approach terminal cell differentiation as a unique system that allows us to combine biochemical, ultrastructural and molecular genetic techniques to study the relationship between the hierarchy of chromatin higher-order structures in the nucleus and its function(s) in dynamic packing of genetic material in a form that remains amenable to regulation of gene activity and other DNA-dependent cellular processes.     

精子染色质结构分析的临床意义

目的探讨女方不明原因流产和不明原因不孕与其配偶精子染色质性状之间的关系;方法通过精子染色质结构分析(SCSA)对22例不明原因流产和36例不明原因不孕妇女配偶精子,以及20例正常对照者精子进行DNA完整性检测,并比较它们之间的差异;结果对照组与不明原因流产组以及不孕组之间精子DNA完整性存在差异,且差异具有统计学意义(P<0．05),而不明原因流产组与不孕组之间精子DNA完整性比较无明显差异(P>0．05);结论精子染色质结构分析有可能作为不明原因流产和不孕的辅助诊断方法在临床应用。     

Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays

BACKGROUND: Sperm DNA integrity (SDI) is an important factor in the prognosis of male fertility. Here we compare the toluidine blue (TB) image cytometry test, recently proposed by us for SDI assessment, with two other tests-the sperm chromatin structure assay (SCSA) and the terminal nick-end labelling (TUNEL) assay. METHODS: Sperm samples from 35 men were evaluated for standard sperm parameters and subjected to the TB test and SCSA. Eighteen of the 35 samples were also subjected to the TUNEL assay. RESULTS: The proportion of sperm cells with abnormal DNA integrity assayed by the TB test correlated strongly with the proportion of abnormal cells detected by the SCSA and TUNEL assay (rho=-0.84 and rho=0.80, P<0.001, respectively). Furthermore, the fractions of abnormal cells by the TB test corresponded closely to the sum of two SCSA parameters, the DNA fragmentation index (DFI) and the fraction of highly DNA-stainable cells (HDS) (medians 33.0 versus 32.0%, P=0.6). CONCLUSIONS: Abnormal cells in a TB test correspond to the sum of DFI and HDS fractions in the SCSA. TB-positive cells may represent sperm with fragmented DNA and/or abnormal chromatin structure. Because the TB test is an easy and inexpensive method, its potential use as a routine test for sperm DNA integrity, complementary to standard semen parameters, should be investigated further.     

Changes in lymphocyte chromatin in Down's syndrome revealed by the thermal denaturation method

A luminescence-microscopic study using acridine orange with a short-term culture of human cells showed that DNA melting profiles of the chromatin of intact lymphocytes of healthy donors are curves with maxima (F 530) in the temperature regions of 45, 65, 78, 85, 88, and 92°C (P<0.01). The melting profiles of lymphocytes of patients with Down's syndrome are curves with maxima in the temperature regions of 65, 85, 88, and 92°C (P<0.01). The absence of a decrease in the intensity of fluorescence between 78 and 85°C is evidently due to the greater degree of condensation of certain regions of the chromatin complex of the trisomic cells. The possible mechanisms of the structural changes in the interphase chromatin, of human lymphocytes under the influence of temperature are discussed.Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Yudaev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 6, pp. 672–674, June, 1976.     

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy

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Regional differences in the compaction of chromatin in human G0/G1 interphase nuclei

The large-scale structure of chromatin corresponding to G- and R-bands in human G₀/G₁ interphase nuclei was compared. Fluorescence in situ hybridization (FISH) was used to measure the interphase distance between 42 pairs of probes separated by 0.1–1.5Mbp. The probe pairs were derived from 21q22.2 and Xp21.3, G-band positive regions, and from 4p16.3, 6p21.3, and Xq28, R-band positive regions. Distributions of measured interphase distances in all regions approximated a Rayleigh distribution, suggesting that the chromatin follows a random-walk path over this range. A linear correlation of mean-square interphase distance and genomic separation, also indicative of random-walk folding, was observed in all regions. The slope of the correlation observed using probes from G-band regions was systematically lower than that from R-band regions. The difference in the slope between Xp21.3 and Xq28 was particularly striking and was observed in normal fibroblast cells, fixed alternatively with methanol and acetic acid or paraformaldehyde, and HeLa cells. These results demonstrate regional differences in large-scale chromosome structure during interphase, with the more openly configured chromatin corresponding to R-bands.This revised version was published online in November 2005 with corrections to the Cover Date.     

Metabolic polymorphisms and the micronucleus frequency in buccal epithelium of adolescents living in an urban environment

Micronuclei and other biomarkers were evaluated in oral cells from 11- to 16-year-old girls living in a foster home in the central area of México City. Variables analyzed for possible association with these biomarkers include smoking habits, body mass index, metabolic polymorphisms for NAT1 and GSTM1 and whether the cells were obtained from the cheek or pharynx. The results indicated that individuals having the NAT1*10 homozygous genotype showed a significant increase in chromatin buds and binucleated cells. When the damage in the cheek was compared with damage in the pharynx, a significant increase in micronuclei and binucleated cells was found for the latter tissue in all the individuals analyzed.     

Reversibility of structural changes in chromatin of interphase nuclei of peripheral blood lymphocytes of patients with Down's syndrome under the influence of healthy human serum

A luminescence-microscopic investigation of short-term human cell cultures stained with Acridine Orange showed that the melting curve of the cell chromatin DNA in the region from 78 to 85°C depends on changing external environmental conditions, i.e., on the composition of the blood serum.Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Yudaev.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 878–879, July, 1976.