Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2.

Entry into mitosis during the somatic cell cycle is regulated in response to signals that monitor the completion of DNA replication, the integrity of the nuclear genome, and, possibly, the increase in cellular mass during the cell cycle. It has been postulated that the operation of this cell cycle control involves the gradual accumulation of rate-limiting mitotic inducers, which trigger nuclear division when their cellular concentration reaches a critical level. We have cloned a human gene, which we call CDC25, whose product may function as a mitotic inducer. This human gene encodes a protein with a predicted molecular mass of 53,000 daltons whose C-terminal domain shares about 37% sequence identity with the fission yeast cdc25+ mitotic inducer. The human CDC25 gene rescues the defect of a fission yeast temperature-sensitive (ts) cdc25ts mutant that is unable to initiate mitosis. In HeLa cells CDC25 mRNA levels are very low in G1 and increase at least 4-fold as cells progress towards M phase. These data suggest that in human cells, as in fission yeast, the accumulation of CDC25 mitotic inducer during G2 may play a key role in regulating the timing of mitosis.     

The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks

The origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae as a protein that specifically binds to origins of DNA replication. Although ORC appears to play an essential role in the initiation of DNA replication in the cells of all eukaryotes, its interactions with DNA have not been defined in species other than budding yeast. We have characterized a Schizosaccharomyces pombe homologue of the ORC subunit, Orc4p. The homologue (Orp4p) consists of two distinct functional domains. The C-terminal domain shows strong sequence similarity to human, frog, and yeast Orc4 proteins, including conserved ATP-binding motifs. The N-terminal domain contains nine copies of the AT-hook motif found in a number of DNA-binding proteins, including the members of the HMG-I(Y) family of chromatin proteins. AT-hook motifs are known from biochemical and structural studies to mediate binding to the minor groove of AT-tracts in DNA. Orp4p is essential for viability of Sc. pombe and is expressed throughout the cell cycle. The Orp4 protein (and its isolated N-terminal domain) binds to the Sc. pombe replication origin, ars1. The DNA binding properties of Orp4p provide a plausible explanation for the characteristic features of Sc. pombe origins of replication, which differ significantly from those of Sa. cerevisiae.     

Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast

A mutation in the Schizosaccharomyces pombe sid4(+) (septation initiation defective) gene was isolated in a screen for mutants defective in cytokinesis. We have cloned sid4(+) and have found that sid4(+) encodes a previously unknown 76.4-kDa protein that localizes to the spindle pole body (SPB) throughout the cell cycle. Sid4p is required for SPB localization of key regulators of septation initiation, including the GTPase Spg1p, the protein kinase Cdc7p, and the GTPase-activating protein Byr4p. An N-terminally truncated Sid4p mutant does not localize to SPBs and when overproduced acts as a dominant-negative mutant by titrating endogenous Sid4p and Spg1p from the SPB. Conversely, the Sid4p N-terminal 153 amino acids are sufficient for SPB localization. Biochemical studies demonstrate that Sid4p interacts with itself, and yeast two-hybrid analysis shows that its self-interaction domain lies within the C-terminal half of the protein. Our data indicate that Sid4p SPB localization is a prerequisite for the execution of the Spg1p signaling cascade.     

CD25+ cell depletion hastens the onset of severe disease in collagen-induced arthritis

OBJECTIVE: CD4+,CD25+ T regulatory cells may offer opportunities to intervene in the course of autoimmune disease. We wished to evaluate their potential for influencing systemic and chronic joint inflammation by investigating their involvement in collagen-induced arthritis (CIA). METHODS: We depleted DBA/1 mice of CD25+ regulatory cells by injection of a depleting monoclonal antibody specific for CD25 14 days before a single immunization with type II collagen (CII) in Freund's complete adjuvant. CD4+,CD25+ T cells were adoptively transferred to some groups of mice during immunization. Mice were then scored for signs of arthritis, and blood was taken periodically to measure the amounts of CII-specific antibodies. Splenocytes of treated mice were examined in vitro to determine the effects of depletion on proliferation to CII and control antigens. RESULTS: CD25+ cell-depleted DBA/1 mice had significantly more severe disease than control mice following collagen immunization. The magnified severity was also accompanied by higher antibody titers against collagen, and in vitro tests showed increased proliferation of collagen-specific T cells. Adoptively transferring CD4+,CD25+ T cells into depleted mice was shown to reverse the heightened severity. Control mice, which were depleted and immunized with the neoantigen keyhole limpet hemocyanin (KLH), had neither an increased antibody response toward KLH nor an augmented proliferative response, indicating that CD25+ cell depletion preferentially affects immunity against self antigen. CONCLUSION: These results establish a link between CD4+,CD25+ regulatory cells and CIA and provide a rationale for investigating CD4+,CD25+ T regulatory cells in the treatment and prevention of arthritis.     

Idiopathic CD4+ T lymphocytopenia disclosed by the onset of empyema thoracis

A 56-year-old man was admitted to our hospital in December 1996 due to empyema thoracis. A laboratory examination revealed lymphocytopenia and CD4+ T lymphocytopenia (<300 cells/ microl). No evidence for a human immunodeficiency virus (HIV) infection was found. No malignant, hematological or autoimmune disease was detected. We thus diagnosed this case as being idiopathic CD4+ T lymphocytopenia (ICL). During his hospital treatment, he was affected with cytomegaloviral retinitis and cured by therapy. His subsequent treatment went well without a recurrence of severe infection although a low CD4+ T lymphocyte count continued after the recovery from empyema thoracis.     

支气管哮喘大鼠CD4+CD25+T细胞的变化及地塞米松干预作用的研究

目的 研究支气管哮喘(简称哮喘)大鼠模型支气管肺泡灌洗液(BALF)、血液、脾脏CD4+CD25+T细胞的变化,及地塞米松对CD4+CD25+T细胞的影响.方法 50只SD大鼠随机分为5组,空白对照(A)组,哮喘(B)组,地塞米松1(C)组、地塞米松2(D)组,地塞米松3(E)组.A组第l天给予腹腔注射生理盐水l ml,第15～21天每天给予生理盐水雾化.B、C、D、E组用卵蛋白建立哮喘大鼠模型,第1天,每只大鼠腹腔注射抗原l ml(卵蛋白1 mg+灭活百日咳杆菌9×106个+氢氧化铝干粉100 mg)混悬液,第15～21天给予1%的卵蛋白雾化30 min,C、D、E组于雾化后分别给予腹腔注射地塞米松0.2 mg/kg、1 mg/kg、2 mg/kg.采用流式细胞仪检测的方法 ,观察大鼠体内BALF、外周血、脾脏CD4+CD25+T细胞的变化及使用不同剂量地塞米松后对其的影响.结果 B组BALF、外周血、脾脏CD4+CD25+T细胞表达占CD4+T细胞的百分比分别是(42.21±5.62)%、(12.69±2.70)%、(11.15±1.05)%,A组结果 分别是(18.76±5.85)%、(6.21±1.73)%、(7.85±2.13)%.B组与A组比较,差异均具有统计学意义(P＜0.01,P＜0.01,P＜0.05);C组、D组、E组BALF中CD4+CD25+T细胞占CD4+T细胞的百分比表达分别是(10.49±4.03)%、(13.28±5.12)%、(7.51±5.39)%,显著低于A组和B组,(P＜0.05,P＜0.01);外周血中,C组(6.03±1.43)%、D组(4.88±0.95)%与A组(6.21±1.73)%比较,差异无统计学意义,E组(3.49±0.62)%与C组、A组比较,差异有统计学意义(P＜0.05).脾脏中,C组(7.25±1.82)%、D组(8.63±3.18)%与A组(7.85±2.13)%比较,差异无统计学意义,E组(3.38±1.37)%与C组、D组、A组比较,差异有统计学意义(P＜0.05).结论 CD4+CD25+T细胞在哮喘大鼠体内有明显的优势表达,可能是哮喘发病的机制之一.地塞米松可以抑制CD4+CD25+T细胞的表达.BALF内CD4+CD25+T细胞的变化与外周血和脾脏的变化具有一致性,监测外周血或脾脏CD4+CD25+T细胞变化可了解肺部情况.     

支气管哮喘大鼠CD4^＋CD25^＋T细胞的变化及地塞米松干预作用的研究

目的研究支气管哮喘（简称哮喘）大鼠模型支气管肺泡灌洗液（BALF）、血液、脾脏CD4^＋CD25^＋T细胞的变化,及地塞米松对CD4^＋CD25^＋T细胞的影响。方法50只SD大鼠随机分为5组,空白对照（A）组,哮喘（B）组,地塞米松1（C）组、地塞米松2（D）组,地塞米松3（E）组。A组第1天给予腹腔注射生理盐水1ml,第15～21天每天给予生理盐水雾化。B、C、D、E组用卵蛋白建立哮喘大鼠模型,第1天,每只大鼠腹腔注射抗原1ml（卵蛋白1mg＋灭活百日咳杆菌9×10。个＋氢氧化铝干粉100mg）混悬液,第15～21天给予1％的卵蛋白雾化30min,C、D、E组于雾化后分别给予腹腔注射地塞米松0．2mg／kg、1mg／kg、2mg／kg。采用流式细胞仪检测的方法,观察大鼠体内BALF、外周血、脾脏CD4^＋CD25^＋T细胞的变化及使用不同剂量地塞米松后对其的影响。结果B组BALF、外周血、脾脏CD4^＋CD25^＋T细胞表达占CD4^＋T细胞的百分比分别是（42．21±5．62）％、（12．69±2．70）％、（11．15±1．05）％,A组结果分别是（18．76±5．85）％、（6．21±1．73）％、（7．85±2．13）％。B组与A组比较,差异均具有统计学意义（P〈0．01,P〈0．01,P〈0．05）;C组、D组、E组BALF中CD4^＋CD25^＋T细胞占CD4^＋T细胞的百分比表达分别是（10．49±4．03）oA、（13．28±5．12）％、（7．51±5．39）％,显著低于A组和B组,（P〈0．05,P〈0．01）;外周血中,C组（6．03±1．43）％、D组（4．88±0．95）％与A组（6．21±1．73）％比较,差异无统计学意义,E组（3．49士0．62）％与C组、A组比较,差异有统计学意义（P〈0．05）。脾脏中,c组（7．25±1．82）%、D组（8．63±3．18）％与A组（7．85±2．13）％比较,差异无统计学意义,E组（3．38±1．37）％与C组、D组、A组比较,差异有统计学意义（P〈0．05）。结论CD4^＋CD25^＋T细胞在哮喘大鼠体内有明显的优势表达,可能是哮喘发病的机制之一。地塞米松可以抑制CD4^＋CD25^＋T细胞的表达。BALF内CD4^＋CD25^＋T细胞的变化与外周血和脾脏的变化具有一致性,监测外周血或脾脏CD4^＋CD25^＋T细胞变化可了解肺部情况。     

Significant competitive advantage conferred by meiosis and syngamy in the yeast Saccharomyces cerevisiae.

The presumed advantages of genetic recombinations are difficult to demonstrate directly. To investigate the effects of recombination and background heterozygosity on competitive ability, we have performed serial-transfer competition experiments between isogenic sexual and asexual strains of the yeast Saccharomyces cerevisiae. The members of these diploid pairs of strains differed only in being heterozygous (sexual) or homozygous (asexual) at the mating type or MAT locus. Competing pairs had either a completely homozygous or a heterozygous genetic background, the latter being heterozygous at many different loci throughout the genome. A round of meiotic recombination (automixis) conferred a large and statistically significant enhancement of competitive ability on sexual strains with a heterozygous genetic background. By contrast, in homozygous background competitions, meiosis decreased the sexual strains' initial relative competitive ability. In all cases, however, the sexual strains outcompeted their isogenic asexual counterparts, whether meiotic recombination had occurred or not. In some genetic backgrounds, this was due in part to an overdominance effect on competitive advantage of heterozygosity at the MAT locus. The advantage of the sexual strains also increased significantly during the course of the homozygous background competitions, particularly when meiosis had occurred. This latter effect either did not occur or was very weak in heterozygous background competitions. Overall, sexual strains with heterozygous genetic backgrounds had a significantly higher initial relative competitive ability than those with homozygous backgrounds. The advantage of mating type heterozygosity in this organism extends far beyond the ability to recombine meiotically.     

Role of the cdc25C phosphatase in G2 arrest induced by nitrogen mustard.

G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.     

Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production

CD4+CD25+ regulatory T (Treg) cells control immunologic tolerance and antitumor immune responses. Therefore, in vivo modification of Treg function by immunosuppressant drugs has broad implications for transplantation biology, autoimmunity, and vaccination strategies. In vivo bioluminescence imaging demonstrated reduced early proliferation of donor-derived luciferase-labeled conventional T cells in animals treated with Treg cells after major histocompatibility complex mismatch bone marrow transplantation. Combining Treg cells with cyclosporine A (CSA), but not rapamycin (RAPA) or mycophenolate mofetil (MMF), suppressed Treg function assessed by increased T-cell proliferation, graft-versus-host disease (GVHD) severity, and reduced survival. Expansion of Treg and FoxP3 expression within this population was lowest in conjunction with CSA, suggesting that calcineurin-dependent interleukin 2 (IL-2) production is critically required for Treg cells in vivo. The functional defect of Treg cells after CSA exposure could be reversed by exogenous IL-2. Further, the Treg plus RAPA combination preserved graft-versus-tumor (GVT) effector function against leukemia cells. Our data indicate that RAPA and MMF rather than CSA preserve function of Treg cells in pathologic immune responses such as GVHD without weakening the GVT effect.     

CD25+ cell depletion hastens the onset of severe disease in collagen‐induced arthritis

Objective

CD4+,CD25+ T regulatory cells may offer opportunities to intervene in the course of autoimmune disease. We wished to evaluate their potential for influencing systemic and chronic joint inflammation by investigating their involvement in collagen‐induced arthritis (CIA).

Methods

We depleted DBA/1 mice of CD25+ regulatory cells by injection of a depleting monoclonal antibody specific for CD25 14 days before a single immunization with type II collagen (CII) in Freund's complete adjuvant. CD4+,CD25+ T cells were adoptively transferred to some groups of mice during immunization. Mice were then scored for signs of arthritis, and blood was taken periodically to measure the amounts of CII‐specific antibodies. Splenocytes of treated mice were examined in vitro to determine the effects of depletion on proliferation to CII and control antigens.

Results

CD25+ cell–depleted DBA/1 mice had significantly more severe disease than control mice following collagen immunization. The magnified severity was also accompanied by higher antibody titers against collagen, and in vitro tests showed increased proliferation of collagen‐specific T cells. Adoptively transferring CD4+,CD25+ T cells into depleted mice was shown to reverse the heightened severity. Control mice, which were depleted and immunized with the neoantigen keyhole limpet hemocyanin (KLH), had neither an increased antibody response toward KLH nor an augmented proliferative response, indicating that CD25+ cell depletion preferentially affects immunity against self antigen.

Conclusion

These results establish a link between CD4+,CD25+ regulatory cells and CIA and provide a rationale for investigating CD4+,CD25+ T regulatory cells in the treatment and prevention of arthritis.

     

Impaired suppression of synovial fluid CD4+CD25− T cells from patients with juvenile idiopathic arthritis by CD4+CD25+ Treg cells

Objective

Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25− effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells.

Methods

The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in ³H‐thymidine proliferation assays. Cytokine levels were analyzed using Bio‐Plex Pro assay.

Results

Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA–DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells.

Conclusion

Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype.

     

CD4+CD25+ T细胞在溃疡性结肠炎中的表达及其临床意义

目的 研究溃疡性结肠炎(UC)患者外周血CD 4CD 25调节性T细胞比例的变化,探讨其在UC病理机制中的意义.方法 选择UC患者33例,对照组20例.用流式细胞仪检测外周血CD 4CD 25T细胞阳性率.用RT-PCR检测外周单个核细胞(PBMC)中Foxp3 mRNA的表达.用酶联免疫吸附试验检测血清中IL-10和TGF-β的浓度.结果 UC患者CD 4CD 25T细胞占CD 4T细胞的比例明显低于对照组(P＜0.01),并且与疾病的活动指数及血沉水平均呈显著负相关(R值分别为-0.660和-0.572,P值均＜0.01).UC患者Foxp3 mRNA的表达也明显低于对照组(P＜0.01).两组患者血清IL-10和TGF-β的浓度比较无明显差异(P＞0.05).结论 UC患者外周血CD 4CD 25 调节性T细胞明显降低,与疾病活动性相关,提示这类细胞可能在UC的病理机制中发挥作用.Foxp3表达降低可能是导致CD 4CD 25 T细胞发育障碍的重要因素.     

Analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

The Schizosaccharomyces pombe centromere-linked genes, LYS1 and CYH1 on chromosome I and TPS13 and RAN1 on chromosome II, have been isolated. The genetic order of these markers with respect to their centromeres was determined to establish relative directionality on the genetic and physical maps. Chromosome walking toward the centromeres reveals a group of repetitive sequences that occur only in the centromere regions of chromosomes I and II and at one other specific location in the S. pombe genome, presumably the centromere of chromosome III. The major class of large repeated sequence elements is 6.4 kilobases (kb) long (repeat K), portions of which occur at least twice on chromosome II and in several tandemly arranged intact copies at another centromeric location. Repeat K in turn contains groups of smaller repeats. Genetic recombination is strongly suppressed in the centromere II region, which contains at least 30 kb of repeated sequences. Centromeric DNA organization is much more complex in fission yeast than has been described in budding yeast (Saccharomyces cerevisiae), possibly because of the larger more condensed nature of the S. pombe chromosomes.     

G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A

Xenopus oocytes, which are arrested in G(2) of meiosis I, contain complexes of cyclin B-cdc2 (M phase-promoting factor) that are kept repressed by inhibitory phosphorylations on cdc2 at Thr-14 and Tyr-15. Progesterone induces a cytoplasmic signaling pathway that leads to activation of cdc25, the phosphatase that removes these phosphorylations, catalyzing entry into M phase. It has been known for 25 years that high levels of cAMP and protein kinase A (PKA) are required to maintain the G(2) arrest and that a drop in PKA activity is required for M phase-promoting factor activation, but no physiological targets of PKA have been identified. We present evidence that cdc25 is a critical target of PKA. (i) In vitro, cdc25 Ser-287 serves as a major site of phosphorylation by PKA, resulting in sequestration by 14-3-3. (ii) Endogenous cdc25 is phosphorylated on Ser-287 in oocytes and dephosphorylated in response to progesterone just before cdc2 dephosphorylation and M-phase entry. (iii) High PKA activity maintains phosphorylation of Ser-287 in vivo, whereas inhibition of PKA by its heat-stable inhibitor (PKI) induces dephosphorylation of Ser-287. (iv) Overexpression of mutant cdc25 (S287A) bypasses the ability of PKA to maintain oocytes in G(2) arrest. These findings argue that cdc25 is a physiologically relevant target of PKA in oocytes. In the early embryonic cell cycles, Ser-287 is phosphorylated during interphase and dephosphorylated just before cdc2 activation and mitotic entry. Thus, in addition to its role in checkpoint arrest, cdc25 Ser-287 serves as a site for regulation during normal, unperturbed cell cycles.