Pseudocysts in trichomonads--new insights

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with anti-tubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.     

Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy

The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.     

Interaction of DBC1 with polyoma small T antigen promotes its degradation and negatively regulates tumorigenesis

Deleted in Breast Cancer 1 (DBC1) is an important metabolic sensor. Previous studies have implicated DBC1 in various cellular functions, notably cell proliferation, apoptosis, histone modification, and adipogenesis. However, current reports about the role of DBC1 in tumorigenesis are controversial and designate DBC1 alternatively as a tumor suppressor or a tumor promoter. In the present study, we report that polyoma small T antigen (PyST) associates with DBC1 in mammalian cells, and this interaction leads to the posttranslational downregulation of DBC1 protein levels. When coexpressed, DBC1 overcomes PyST-induced mitotic arrest and promotes the exit of cells from mitosis. Using both transient and stable modes of PyST expression, we also show that cellular DBC1 is subjected to degradation by LKB1, a tumor suppressor and cellular energy sensor kinase, in an AMP kinase-independent manner. Moreover, LKB1 negatively regulates the phosphorylation as well as activity of the prosurvival kinase AKT1 through DBC1 and its downstream pseudokinase substrate, Tribbles 3 (TRB3). Using both transient transfection and stable cell line approaches as well as soft agar assay, we demonstrate that DBC1 has oncogenic potential. In conclusion, our study provides insight into a novel signaling axis that connects LKB1, DBC1, TRB3, and AKT1. We propose that the LKB1–DBC1–AKT1 signaling paradigm may have an important role in the regulation of cell cycle and apoptosis and consequently tumorigenesis.     

Variations in DNA Synthesis and Mitotic Indices in Hepatocytes and Sinusoid Litoral Cells of Adult Intact Male Mouse Along a Orcadian Time Span

Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.     

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies.     

RAS蛋白在有丝分裂信号转导中的作用

致癌基因ｒａｓ编码的ＲＡＳ蛋白参与了多种细胞因子调控的有丝分裂过程。抗ＲＡＳ抗体能抑制细胞正常的有丝分裂。ｒａｓ基因转化的成纤维细胞ＮＩＨ３Ｔ３还具有在无血清和生长因子刺激下自发完成细胞周期的能力。在这种细胞中有活性氧的生成,抗氧化剂能抑制该细胞的有丝分裂。ＲＡＳ蛋白在有丝分裂的信号转导中参与了多条途径,是信号转导的重要组织者     

Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.     

Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

Vesicular stomatitis virus (VSV) infects and kills a wide range of cell types; however, the mechanisms involved in VSV‐mediated cell death are not fully understood. Here we show that VSV infection interferes with mitotic progression, resulting in cell death. This effect requires the interaction of VSV matrix (M) protein with the Rae1–Nup98 complex in mitosis, which is associated with a subset of ribonucleoproteins (RNPs). VSV displaced Rae1 from spindle poles, caused spindle abnormalities and triggered substantial cell death during metaphase. These effects were attenuated in cells infected with VSV expressing a mutant M protein that does not bind efficiently to the Rae1–Nup98–RNP complex. In cells that progressed to late mitosis, M protein prevented proper nuclear formation and chromatin decondensation. VSV is an oncolytic (anti‐tumour) agent as it preferentially replicates and kills tumour cells. As tumour cells have a high mitotic index, VSV‐mediated mitotic cell death probably contributes to its oncolytic activity.     

ULTRASTRUCTURE AND PIGMENTS OF TWO STRAINS OF THE PICOPLANKTONIC ALGA PELAGOCOCCUS SUBVIRIDIS (CHRYSOPHYCEAE)1

The organelle ultrastructure and photosynthetic pigments of a new isolate of the picoplanktonic alga Pelagococcus subviridis Norris from the East Australian Current was compared with the North Pacific Ocean type species. No differences in the ultrastructure of the two isolates were observed. Mitosis was studied in detail in the Australian strain, and showed two unusual features: the de novo appearance of centrioles prior to mitosis, and the formation of a small, extra-nuclear spindle. The major carotenoids in both strains were fucoxanthin and a 19′-butanoyloxyfucoxanthin-like pigment, with diadinoxanthin and diatoxanthin as secondary pigments. Several minor carotenoids have not yet been identified. In addition to chlorophylls a and c₂, a new chlorophyll c derivative (chlorophyll c₃), present in both strains, was separated by high-performance thin-layer chromatography (HPTLC). The Australian isolate, unlike the type material, showed no evidence of chlorophyllase activity during cell harvest and extraction. While the pigment composition suggests affinities with certain newly examined prymnesiophytes, organelle ultrastructure indicates Pelagococcus to be a member of the Chrysophyceae. Mitosis is, however, atypical of both Prymnesiophyceae and Chrysophyceae, and if this picoplanktonic alga is to be retained in the Chrysophyceae it must be seen as a most unusual member.