Kre29p is a novel nuclear protein involved in DNA repair and mitotic fidelity in Candida glabrata

Candida glabrata KRE29 is an ortholog of Saccharomyces cerevisiae KRE29. S. cerevisiae Kre29p has been identified by affinity purification as a subunit of the Smc5–Smc6 complex, which is required for DNA repair and chromosome segregation. However, mutant phenotypes of S. cerevisiae KRE29 have not been well characterized and none of its orthologs’ functions has been reported. Here we report phenotypic characteristics of a C. glabrata kre29 deletant. The absence of C. glabrata Kre29p resulted in decreased viability, exhibiting cell cycle arrest between late S-phase and metaphase even under normal growth conditions, and also caused an increase of plasmid loss rate, implying that Kre29p is required for mitotic chromosome transmission fidelity. The deletant showed increased sensitivity to high temperature as well as to DNA damaging agents including UV, gamma ray, 4-nitroquinoline-1-oxide and methyl methanesulfonate, and the phenotypes were restored in the KRE29 reintegrant. Consistent with the Δkre29 phenotypes, a Kre29p-GFP fusion protein was located in the nucleus. Furthermore, Kre29p-GFP became concentrated and formed distinct foci after exposure to 4-nitroquinoline-1-oxide. These results suggest the involvement of C. glabrata Kre29p in DNA repair. To our knowledge, this is the first report addressing a cellular protein involved in DNA repair in C. glabrata.     

Induction of polyploidy in Adenovirus E1-transformed cells by the mitotic inhibitor colcemid

Adenovirus-transformed cells were tested for their ability to synthesize DNA in the presence of cell cycle inhibitory drugs. We show that transformed cells are completely resistant to the mitotic inhibitor colcemid, partly resistant to lovastatin, mimosine, aphidicolin and genistein but not to hydroxyurea or thymidine. When treated with colcemid, AdE1-transformed cells continue to synthesize DNA but do not divide and, therefore, become highly polyploid. This effect is dependent on the presence of both E1A and E1B.     

Mast cells and postnatal topographic anomalies in mammalian subfornical body and supraoptic crest

Summary In the central nervous system of 22 animal species, mast cells occur (1) in both the subfornical body and the supraoptic crest in chimpanzee, stumpatailed monkey, agouti and flying squirrel and in cynomolgus monkey from 1 day of age; (2) in the subfornical body in paca, ground squirrel, woodchuck and prairie dog; and (3) in the supraoptic crest in cats from 2 days of age through the fourth month, and capybara. The medial habenular nucleus and the area postrema contain mast cells in many of the animals, but differences in number with species and with age do not follow the same pattern as in the above regions. Not infrequently, the thalamus has a considerable number of mast cells, and occasionally other regions, such as the dentate nuclei and the medulla oblongata, have a few mast cells.The number of mast cells, amounting to several hundreds in the circumventricular regions, varies with region, species, individual animal and age. The marked qualitative and quantitative differences, which complicate interpretation of experimental results, are associated with differences in functional requirements; the migratory faculty of the mast cell would enable each of its many biogenic amines to act separately on specific elements at different sites as demand arises.Study of newborn animals disclosed mitotic cells in several of the circumventricular regions. The formation of dysmitotic elements, as an expression of anomalous mitosis, may be the cause of disproportionate growth of the tissue and the overlying ependyma, whereby aberrations in the development of the ventricular wall ensue.     

The REC46 gene of Saccharomyces cerevisiae controls mitotic chromosomal stability,recombination and sporulation: cell-type and life cycle stage-specific expression of the rec46-1 mutation

Summary The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT n+1 chromosome VII disomic strain LBW (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATaIMaT ree46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intergenic recombination resulting in prototrophy. When compared to MAT rec46-1 n+1 disomics however, MATa/MAT rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATaIMAT rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).     

Mitosis and cell death in the tail of the chick embryo

Summary Although somites develop from the mesoderm in the tail of the chick embryo, they do not form to the tip of the tail. Previous work has shown that this terminal mesoderm possesses many of the characteristics of the segmental plate mesoderm which gives rise to the somites in the trunk. This investigation is aimed therefore at understanding why the terminal mesoderm fails to form somites.Mitotic and pyknotic rates have been obtained for the tail region of chick embryos between stages 13 and 27. Embryos were treated with colchicine, so that the mitoses were blocked in metaphase, and counts were made on serial sections. The overall mitotic rates were highest between stages 15 and 18. Regions of high mitotic rate, which are an indication of cell synchrony, were found in the tail bud mesoderm though not in a consistent location, and only infrequently ner the anterior end of the tail segmental plate. In the trunk however (Stern and Bellairs 1984) a single peak of cell synchrony was routinely found near the cranial end of the segmental plate. It is concluded that the cells of the tail mesoderm are less synchronised in preparation for somitogenesis than are the corresponding mesoderm cells in the trunk. A further conclusion is that the tail bud is not per se a region of high proliferation, though there are patches of high mitotic rate.The overall pyknotic rate reached a maximum at stage 25; peaks of pyknosis corresponded initially with the mitotic peaks and were associated with the ventral ectodermal ridge and the tail gut. By stage 25 however, the high levels of cell death were restricted mainly to the tip of the tail. It is concluded that cell death radually predominates over mitosis in the region of prospective somitogenesis. It is suggested moreover that the cells in the ventral ectodermal ridge may play a role in pattern formation similar to that of the apical ectodermal ridge in the limb.Tail mesoderm was dissected from embryos at stage 13 (prior to cell death), and stage 16 (when patches of cell death were already established), and explanted in vitro. The cumulative cell death which occurred within the tissue from stage 13 was significantly less that from stage 16. It is concluded that the cells destined for death may be at least partially rescued if isolated in vitro.     

Effect of α-difluoromethylornithine on the polyamine levels and proliferation in two transplantable tumours

Summary The effect of inhibition of polyamine biosynthesis by-difluoromethylornithine (DFMO) on the growth of two murine transplantable tumours was studied. Female CBA mice were implanted with either the sarcoma F (SaF) or an anaplastic mammary carcinoma (CaNT), and 3% DFMO in the drinking water was provided once the tumours were established. Over a 10-day period control SaF tumours increased exponentially from 20 mm³ to over 800 mm³, whereas DFMO-treated SaF reached only 300 mm³. CaNT grew more slowly, requiring 22 days to achieve a similar volume increase, and DFMO was as effective in retarding growth as it had been in SaF. DFMO depleted tumour tissues of putrescine and spermidine, but did not reduce spermine levels. Metaphase arrest experiments with vincristine demonstrated that DFMO could substantially reduce the rates of tumour cell production, but there was no indication the DFMO accelerated the rate of cell loss from the tumours. Despite reduced rates of cell production, labelling studies with bromodeoxyuridine failed to detect differences between control and treated tumours: an increase in transit time through the S-phase was suspected. The number of nuclear organizer regions, detected by the argyrophilia of their associated proteins, was less in DFMO-treated tumours, and within a tumour the degree of silver deposition unequivocally reflected the proliferative heterogeneity. Ultrastructural studies revealed no differences between DFMO-treated and untreated tumours.     

肝外胆管癌患者癌组织与血清中PLK1、Aurora A水平的变化及其临床意义

目的:探讨有丝分裂调控酶polo样激酶1(PLK1)和Aurora A在肝外胆管癌患者癌组织及血清中的水平以及两者的临床意义。方法:用免疫组化法测定54例肝外胆管癌组织及20例癌旁胆管组织中PLK1和Aurora A的表达,用ELISA法检测25例肝外胆管癌患者与15例健康人血清中PLK1和Aurora A的浓度。分析两者表达与患者临床病理因素的关系,以及患者手术前后两者水平的变化。结果:肝外胆管癌患者中,癌组织中PLK1和Aurora A的阳性表达率均明显高于癌旁胆管组织(66.7%vs.25.0%;63.0%vs.15.0%,均P0.05),且PLK1和Aurora A的表达与肿瘤的分化程度、TNM分期和淋巴结转移密切有关(均P0.05);术前血清中PLK1和Aurora A的浓度均明显高于健康人群(434.85 pg/mL vs.256.00 pg/m L;644.64 pg/m L vs.375.73 pg/m L,均P0.05),术后两者水平均明显降低(均P0.05);PLK1与Aurora A在肝外胆管癌患者癌组织中的表达量与血清中浓度具有一致性(r=0.55;r=0.64,均P0.05);无论癌组织或血清中,PLK1与Aurora A水平均呈正相关(癌组织:r=0.47,P0.01;血清:r=0.71,P0.01)。结论:PLK1与Aurora A水平在肝外胆管癌患者癌组织与血清均升高,且两者水平的升高与肝外胆管癌的恶性进展密切相关;两者联合检测对肝外胆管癌早期诊断、治疗效果及预后的判断有一定价值。     

体外培养状态下心肌细胞有丝分裂的证据

目的探讨体外培养状态下心肌细胞有丝分裂、增殖的证据。方法分离新生大鼠心肌细胞,分别原代培养0h至12d;免疫荧光分别双标心肌肌钙蛋白-T(c Tn T)、磷酸化组蛋白H3(H3P)和微管蛋白(tubulin),利用活细胞工作站和荧光显微镜观察处于分裂期的心肌细胞。结果原代培养的心肌细胞生长状态好、纯度高,可观察到分裂各期的心肌细胞及其微管蛋白的变化。处于分裂各期的心肌细胞不隆起变圆仍维持扁平状。分裂前期细胞核形状规则,染色质凝集,H3P开始表达;前中期核膜破裂,纺锤体形成;中期染色体排列在赤道面,纺锤体呈典型纺锤样;后期染色单体分开并移向两极;末期子核形成,平行微管聚结在两子核之间;胞质分裂期形成两个子细胞,两个细胞分开后,可见有少量微管呈丝状连接。原代培养1~12d的心肌细胞在整个细胞分裂图像中很少见到双核心肌细胞。培养第3天开始发现分裂象的心肌细胞,占总心肌细胞数的2/127(1.57%)和1/92(1.09%),培养第7天,分裂细胞比率达3/69(4.35%)和5/163(3.07%),培养第12天有2/100(2%)和0/60(0)的心肌细胞处于分裂期。结论体外培养的心肌细胞存在一定数量的完全有丝分裂,是心肌增殖和心肌再生的细胞学证据。     

内皮素1对喉癌细胞HEP 2的促增殖作用

目的探讨内皮素1对HEP 2细胞有丝分裂的影响。方法采用RT PCR及免疫组化Dako EnVision法检测HEP 2细胞内皮素受体表达情况;3H 胸腺嘧啶核苷渗入法检测不同浓度和作用时间内皮素1对HEP 2细胞有丝分裂的影响。结果HEP 2细胞存在内皮素受体基因及蛋白表达;内皮素1具有明显促HEP2细胞增殖作用,且其促增殖作用呈浓度依赖性。结论内皮素1能有效促进HEP 2细胞有丝分裂,对喉癌的演进可能有重要调控作用。