Technique for the visualization of exchange aberrations in human chromosomes.

A technique for the visualization of chromatid exchange in human chromosomes is described. Additon of the base analogue 5-bromodeoxyuridine (BUdR) into PHA stimulated lymphocyte cultures and subsequent staining with the benzimidazol compound "33258 Hoechst", and Giemsa, results in differential fluorescence or staining of sister chromatids in second division metaphases. This technique allows an accurate scoring of sister chromatid exchanges, and could be used in the evaluation of the effect of external agents on the induction of previously unrecognized chromatid exchanges in human chromosomes.     

Early detection of Chlamydia trachomatis using fluorescent, DNA binding dyes.

HeLa 229 cells were infected with genital tract strains of Chlamydia trachomatis. After incubation for varying times the infected cells were fixed and stained with the fluorescent DNA binding dyes Hoechst 33258 or DAPI for comparison with conventional Giemsa stain. Fluorochrome-treated preparations were examined by incident ultraviolet fluorescence microscopy and the Giemsa-stained preparations by dark-ground light microscopy. Chlamydial inclusion bodies could be identified unambiguously as early as 18 hours after infection of HeLa 229 cells using either Hoechst 33258 or DAPI but not until some 48 hours in Giemsa-stained preparations. The DNA rich chlamydial elementary bodies in infected egg yolk suspension were readily detected using Hoechst 33258. The fluorescent dye technique was simpler and more rapid than Giemsa staining. Using Hoechst 33258 it is possible to speed up the identification of chlamydial isolates growing in tissue culture.     

Detection of parasites with DNA-binding bisbenzimide H33258 in Pneumocystis carinii - and Leishmania -containing materials

Even for routine purposes, standard staining of Pneumocystis- or Leishmania-containing materials, e.g., with Giemsa or Diff-Quik, is often unsatisfactory due to poor contrast and to staining of irrelevant structures. In comparison, the bisbenzimide dye Hoechst 33258, a DNA-binding fluorochrome, allows a more precise analysis of such materials. Bisbenzimide stained all stages of these fungal or protozoal organisms with brilliant contrast against a uniformly dark background. The level of background luminescence and staining of detritus or non-DNA structures was very low. Organisms were stained both outside of and within phagocytic cells with equal intensity. Counting of individual microorganisms, e.g., in macrophages heavily parasitized with Leishmania or in Pneumocystis-infected bronchoalveolar lavage, was simplified and more precise. Air-dried cell suspensions, cytocentrifuge preparations, impression smears, or cryocut micrographs showed the advantages of bisbenzimide staining over Diff-Quik. Staining with bisbenzimide could be a valuable auxiliary technique for the analysis of material infected with a variety of microorganisms. Received: 29 November 1997 / Accepted: 5 January 1998     

Hoechst 33342 induces apoptosis in HL-60 cells and inhibits topoisomerase I in vivo.

CONTEXT: Bisbenzimides (Hoechst 33342 and Hoechst 33258) are cell-permeable, adenine-thymine-specific dyes that bind to the minor groove of DNA and stain DNA. Hoechst 33342 induces apoptosis in BC3H-1 myocytes and hepatoma cells. OBJECTIVE: To determine if Hoechst 33342 or Hoechst 33258 induces apoptosis in human promyelocytic leukemia cells (HL-60) and inhibits topoisomerase I activity. DESIGN: A variety of methods were used to detect apoptosis: cell viability (trypan blue exclusion), nuclear fluorescence staining (Hoechst 33342 or Hoechst 33258 stained for 10 minutes), flow cytometric quantitation of annexin binding to phosphatidylserine, and DNA fragmentation (agarose gel electrophoresis). Topoisomerase I activity was determined by a plasmid unwinding assay. SETTING: A large teaching hospital and research laboratories. PATIENTS: None. INTERVENTION: None. MAIN OUTCOME MEASUREMENTS: Apoptosis is characterized by decreased cell viability, condensation of nuclear chromatin, increased phosphatidylserine translocation, and DNA fragmentation into oligonucleosomes composed of multiples of 180 to 200 base pairs. Inhibition of endogenous nuclear topoisomerase I is detected by the absence of plasmid unwinding from a tightly coiled to relaxed form. RESULTS: Hoechst 33342, but not Hoechst 33258, induced apoptosis in the HL-60 cells in a time- and dose-dependent manner. Endogenous nuclear topoisomerase I activity in HL-60 cells was inhibited by treatment with Hoechst 33342 but not Hoechst 33258. CONCLUSION: Hoechst 33342-induced HL-60 cell apoptosis may be related to the dye's inhibition of topoisomerase I activity.     

Simultaneous detection of SCE and Q-bands on human chromosomes by a double-staining technique

Human lymphocytes were cultured for two cell cycles in the presence of bromodeoxyuridine (BrdU), and the resulting metaphase chromosomes were first stained with quinacrine mustard (QM) and then, immediately afterwards, with Hoechst 33258, without any intermediate destaining. Both Q-banding patterns and sister chromatid differential staining were photographed subsequently on the same metaphase using two different filter blocks of the fluorescence microscope.     

Bromodeoxyuridine tablet methodology for in vivo

5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.     

Hoechst 33342-induced apoptosis is associated with decreased immunoreactive topoisomerase I and topoisomerase I-DNA complex formation

Hoechst 33342, but not Hoechst 33258, induces apoptosis and inhibits topoisomerase 1 activity in vivo. Topoisomerase I relaxes superhelical DNA through a single strand breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site, forming a transient intermediate called a cleavable complex. The fate of cellular topoisomerase 1 in Hoechst 33342-induced apoptosis is unknown. We analyzed the binding capacity of topoisomerase 1 to 32P-labeled plasmid pCI DNA, the immunoreactive topoisomerase 1 concentration and topoisomerase 1 activity in BC3H-1 myocytes and HL-60 cells treated with Hoechst 33342 and Hoechst 33258 by using covalent transfer of 32P radioactivity from plasmid DNA to topoisomerase 1, Western blotting and topoisomerase 1-mediated plasmid relaxation assay, respectively. Hoechst 33342, but not Hoechst 33258, induced topoisomerase 1 dysfunction in both BC3H-1 myocytes and HL-60 cells measured by (1) a decrease in the topoisomerase 1 to DNA binding capacity or cleavable complex formation; (2) a decrease in intracellular concentration of immunoreactive topoisomerase 1; and (3) an inhibition of nuclear endogenous topoisomerase 1 activity. These results suggest that destruction of immunoreactive topoisomerase 1 and topoisomerase 1-DNA complexes or cleavable complexes results in inhibition of topoisomerase 1 activity, a key step in the Hoechst 33342-induced apoptotic process.     

幽门螺杆菌对胃上皮细胞SGC-7901凋亡的影响

目的：观察幽门螺杆菌（H. pylori）能否诱导体外培养的胃上皮细胞发生凋亡。方法：采用荧光染色、流式细胞仪和透射电子显微镜技术,观察H. pylori活菌在体外对人胃腺癌细胞系SGC-791凋亡形态与凋亡率的影响。结果：Hoechst 33258荧光染色和流式细胞术两种方法所得结果一致,3.2×10⁷ CFU/L的H. pylori组与对照组的细胞凋亡率无显著差异（P>0.05）,当H. pylori的浓度1.6×10⁸ CFU/L时,随着H. pylori浓度的加大,细胞凋亡率越来越高,均与对照组有显著差异（P<0.01）;细胞110CFU/L的H. pylori共育48 h后,在透射电镜下可见到部分细胞呈现凋亡形态：核中染色质不规则凝集,核膜向多处伸出指状突起,另外还可见胞浆浓缩成均质状,可形成胞浆凋亡小体。结论：H. pylori活菌能以浓度依赖方式诱导体外培养的SGC-791发生凋亡。     

Sister chromatid differentiation in 5-bromo-2′-deoxyuridine-substituted chromosomes: A study with DNA-specific ligands and monoclonal antibody to histone H2B

Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol—acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double-or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B. Our data exclude the possibility that the presence/absence of BrdU in the template strand might affect chromatin organization and thus resistance, while confirming that UV-induced DNA alteration is not sufficient, by itself, to explain SCD mechanism(s). That molecules other than DNA play a role in explaining SCD production is indicated by the fact that BrdU incorporation induces alterations in DNA—histone H2B interactions which, in turn, seem to produce structural variations in chromatin, possibly at the level of condensation, as monitored by phase-contrast and electron microscopy.     

P>三氮唑席夫碱衍生物诱导SMMC-7721细胞分化为多倍体巨细胞/P>

目的 探讨三氮唑席夫碱衍生物（LH-37）诱导SMMC-7721细胞形成多倍体巨细胞的作用。方法 SMMC-7721细胞以1×10⁴/ml的浓度接种于含1×10^-5 mol/L LH-37的培养液内72h,流式细胞术分析DNA含量,Hoechst33258和碘化丙啶双染观察细胞死亡,免疫荧光检查微管和核纤层构造,透射电镜观察巨细胞超微结构。结果 LH-37 作用SMMC-7721细胞72h,巨细胞明显增加,单核巨细胞和多核巨细胞分别为12.32%和0.92%。可见多核巨细胞子核放射状排列,微管增厚并包裹子核,形成单一的微管组织中心,放射状排列的子核被完整的核纤层分割包绕,大部分多核巨细胞凋亡。单核巨细胞的微管常围绕核膜凝集,同时向细胞膜方向放射状分布,可见异常的有丝分裂和凝集的染色质,电镜下可见一些单核巨细胞凋亡时伴有自吞噬特征。结论 LH-37通过干扰微管动力学,导致多倍体巨细胞增加和延迟性细胞死亡。     

Heritable fragile sites and cancer: fra(16)(q22) in lymphocytes of an acute nonlymphocytic leukemia patient with inv(16)(p13q22)

Fragile site testing was performed on normal peripheral blood lymphocytes from three acute nonlymphocytic leukemia patients who carried inv(16)(p13q22) in malignant cells. Cultures were treated with BrdU, distamycin A, Hoechst 33258, or folic acid deprivation to induce fragile site expression. One patient was found to be a carrier of fra(16)(q22), but the expression was observed only by Hoechst 33258 treatment.     

Effect of sperm-antibodies on acrosome reaction of human sperm used for the hamster egg penetration assay

The effect of anti-sperm antibodies (ASA) on the rate of acrosome reactions (AR) during "in vitro" capacitation of human sperm used for the hamster egg penetration assay (HEPA) was assessed. Motile sperm suspensions from donors were exposed to several sera and seminal plasma with sperm head-directed ASA, then they were washed and capacitated "in vitro." After capacitation, the proportion of acrosome-reacted viable sperm was assessed by staining with Fluoresceinated Pisum Sativum Agglutinin and supravital stain Hoechst 33258. ASA of any immunoglobulin class did not significantly affect either the AR rate, or the hamster egg penetration rate. In conclusion, interference of ASA on spontaneous AR rate during "in vitro" capacitation can not be advocated as an explanation of the impairment of the interaction of human sperm with egg or its vestments, which have been reported in several studies.     

冬凌草甲素增强巨噬细胞对凋亡的U937细胞的吞噬作用

目的：研究具有诱导肿瘤细胞凋亡活性的冬凌草甲素，促进巨噬细胞对因凋亡的肿瘤细胞的吞噬作用。 方法： DNA凝胶电泳检测UV照射（2.4 J/cm2, 4 min）的人组织淋巴瘤U937细胞凋亡；Giemsa染色，Hoechst 33258染色，镜下检测计数吞噬作用。 结果： UV照射（2.4 J/cm2, 4 min）诱导U937细胞发生凋亡，琼脂糖凝胶电泳可见凋亡典型的DNA梯带。2.7 μmol·L^-1的冬凌草甲素具有增强U937分化的巨噬细胞对UV照射诱导凋亡的U937细胞的吞噬作用，并呈时间剂量依赖性，但对非特异性荧光颗粒的吞噬效果较弱。加入抗TNFα和抗IL-1β的抗体，培养12 h, 吞噬增强作用明显受抑制。冬凌草甲素在人外周血来源的巨噬细胞吞噬凋亡的U937细胞过程中同样发挥增强吞噬的效果。 结论： 冬凌草甲素可特异地增强巨噬细胞对凋亡的U937细胞的吞噬作用，其吞噬机制是通过诱导巨噬细胞TNFα和IL-1β的释放。     

姜黄素诱导永生化人HaCaT细胞凋亡

目的 探讨姜黄素对人永生化上皮细胞凋亡的诱导作用.方法 应用细胞计数、流式细胞术、琼脂糖凝胶电泳、Hoechst33258染色、苏木素-伊红(HE)染色和透射电镜检测经姜黄素诱导处理后人永生化上皮HaCaT细胞的凋亡.结果经7.5mg/L姜黄素诱导处理后,人永生化上皮HaCaT细胞的增殖活动受到明显的抑制,细胞生长抑制率达83.03%;细胞周期检测出现亚二倍体(亚G1期)细胞峰值,细胞凋亡率达13.1%,并发生G2/M期阻滞;琼脂糖凝胶电泳出现细胞凋亡典型的DNA"梯状"条带;细胞核经Hoechst33258染色出现浓染致密的固缩形态和颗粒状荧光;光镜和电镜观察结果显示,姜黄素处理组细胞体积缩小,细胞核固缩,染色质凝聚,线粒体肿胀,有凋亡小体形成等显著的凋亡特征.结论姜黄素对人永生化上皮HaCaT细胞凋亡有显著的诱导作用,从而为进一步研究表皮细胞衰老、凋亡机理提供了重要基础和研究依据.     

Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa.

Fluorescein-conjugated peanut agglutinin (PNA) lectin-labelling is an established procedure for assessing the status of the human sperm acrosome. However, unlike the triple-stain technique, PNA-labelling does not provide a simultaneous assessment of cellular vitality. We have therefore evaluated the use of the fluorescent dye Hoechst 33258 (H33258) as a vital stain for use in combination with PNA-labelling. Human sperm populations were stained for 1 min with 1 microgram/ml H33258 in culture medium and then washed through 2.0 (w/v) polyvinylpyrollidone columns and air-dried onto microscope slides. H33258 was found to provide vitality assessments comparable to those obtained using the standard eosin-exclusion method. However, best results were obtained with an ethanol fixation step between air-drying and PNA-labelling. This vitality assessment was found to be more reliable than that provided by Trypan blue staining under conditions equivalent to the triple-stain technique. There was no alteration of PNA-labelling due to the H33258 although ethanol fixation actually provided more uniform PNA-labelling than previously obtained without ethanol fixation. Consequently, we have stopped using the triple-stain technique for assessing human sperm acrosome reactions and now use the H33258/PNA procedure routinely.     

Analysis of crystallite shape in rat incisor enamel

The hydroxyapatite crystallites of mammalian enamel appear as hexagons when seen in cross-sections examined with the transmission electron microscope. Using goniometric transmission electron microscopy, stereo-pair electron micrographs and freeze-fracture replicas, two models have been proposed to explain the hexagonal crystallite profile. The "hexagonal ribbon" model proposes that hexagonal profiles are true cross-sections of elongated hexagonal ribbons. The "rectangular ribbon"model proposes that crystallite profiles are three-dimensional rectangular segments (parallelepipeds), which are contained in the Epon sections and project as opaque hexagons in routine transmission electron micrographs. Morphological observations together with predictions from models indicate that the crystallites in rat incisor enamel are flat ribbons with rectangular cross-sectional profiles. The hexagonal images seen in electron micrographs of thin sections of enamel result from viewing parallelepiped-shaped segments of these crystallites as two-dimensional shadows.     

Sequence of centromere separation: kinetochore formation in induced laggards and micronuclei

Mouse L-cells were treated with bis-benzimidazole derivative (Hoechst 33258), caffeine and bleomycin in order to study genesis of laggards and micronuclei and formation of kinetochores as revealed by antikinetochore antibody staining. Apparently, the Hoechst 33258-induced decondensation experienced by the A:T-rich pericentric heterochromatin does not extend into the centromeric region and does not affect formation, physical appearance or function of kinetochores. The laggards induced by Hoechst 33258 are generally whole chromosome laggards which have antikinetochore antibody binding sites. These kinetochore-carrying laggards were seen to lie outside the spindle region in cells untreated with spindle inhibitors or hypotonic and stained differentially for spindle and chromosomes. Some micronuclei did not show kinetochore dots indicating their origin in acentric chromosome fragments. When cells were treated with caffeine or bleomycin, both types of micronuclei, namely those carrying kinetochores and those generated by acentric fragments, were seen. These results are interesting in that caffeine prevents the rejoining of chromosome breaks and one would expect only kinetochore-less micronuclei in caffeine-treated cells. It may mean that caffeine also induces aneuploidy. The L-cells carry minichromosomes which are no more than a pair of kinetochore dots. Such chromosomes, though detectable by antikinetochore antibody staining, may be missed in routine, acid-fixed, Giemsa-stained preparations.     

CD71和Hoechst33258用于孕妇外周血中胎儿有核红细胞的分选

目的 比较ＣＤ７１单染和ＣＤ７１、Ｈｏｅｃｈｓｔ３３２５８（ＨＯ２５８）双染对单个核细胞的标记情况,并用于对孕妇外周血中有核红细胞（ｎｕｃｌｅａｔｅｄ ｒｅｄ ｂｌｏｏｄ ｃｅｌｌｓ,ＮＲＢＣｓ）的分选。方法 选用红细胞特异性抗体ＣＤ７１、核染料ＨＯ２５８对正常孕妇外周血、轻（或）中度妊娠高血压综合征（简称妊高症）患者外周血、初生婴儿脐血单个核细胞进行标记,并结合流式细胞术对有核红细胞进行分选。结     

Oridonin-induced apoptosis of Jurkat cells and its mechanism

Jurkat cells in culture medium in vitro were exposed to different concentrations of oridonin. The proliferation rate of the cells was measured by MTT assay, cell apoptotic rate was detected by flow cytometry, morphology of cell apoptosis was observed by Hoechst 33258 fluorescence staining, DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3 and poly(ADP-ribose) polymerase (PARP) expressions were detected by Western blotting using polyclonal anti-caspase-3 antibody and mouse anti-human PARP monoclonal antibodies, and caspase-3 activity was assayed with a colorimetric assay kit before and after apoptosis had occurred. Oridonin (over 32 mol/l) inhibited the growth of Jurkat cells and caused significant apoptosis. The suppression was both time-dependent and dose-dependent. Marked morphological changes of cell apoptosis, including condensation of chromatin and nuclear fragmentation, were clearly observed by Hoechst 33258 fluorescence staining, as well as by agarose gel electrophoresis. Western blotting showed cleavage of the caspase-3 zymogen protein (32 kDa), with the appearance of its 17 kDa subunit, and a cleaved 89-kDa fragment of 116 kDa PARP was also found, together with a concurrent increase in caspase-3 apoptotic activity. Oridonin can induce apoptosis in Jurkat cells via activation of caspase-3; the results indicate that oridonin might be an important potential anti-leukaemia reagent.