The Ndc80 outer kinetochore complex plays a critical role in kinetochore-microtubule attachment, yet our understanding of the mechanism by which this complex interacts with spindle microtubules for ...timely and accurate chromosome segregation remains limited. Here we address this issue using an ndc80 mutant (ndc80-NH12) from fission yeast that contains a point mutation within a ubiquitous internal loop. This mutant is normal for assembly of the Ndc80 complex and bipolar spindle formation yet defective in proper end-on attachment to the spindle microtubule, with chromosome alignment defects and missegregation happening later during mitosis. We find that ndc80-NH12 exhibits impaired localization of the microtubule-associated protein complex Alp7/transforming acidic coiled coil (TACC)-Alp14/tumor-overexpressed gene (TOG) to the mitotic kinetochore. Consistently, wild-type Ndc80 binds these two proteins, whereas the Ndc80-NH12 mutant protein displays a substantial reduction of interaction. Crucially, forced targeting of Alp7-Alp14 to the outer kinetochore rescues ndc80-NH12-mutant phenotypes. The loop was previously shown to bind Dis1/TOG, by which it ensures initial chromosome capture during early mitosis. Strikingly, ndc80-NH12 is normal in Dis1 localization. Genetic results indicate that the loop recruits Dis1/TOG and Alp7/TACC-Alp14/TOG independently. Our work therefore establishes that the Ndc80 loop plays sequential roles in spindle-kinetochore attachment by connecting the Ndc80 complex to Dis1/TOG and Alp7/TACC-Alp14/TOG.
Background
It was recently reported that genetic polymorphisms of UDP glucuronyltransferase-1 polypeptide A1 (UGT1A1), a glucuronidation enzyme, were associated with irinotecan (CPT-11) metabolism. ...The active metabolite of CPT-11, 7-ethyl-10-hydroxycamptothecin (SN-38) was glucuronidated (SN-38G) by UGT1A1. Genetic polymorphisms of UGT1A1 were associated with potentially serious adverse events, including neutropenia. Several studies have suggested that the dose of CPT-11 should be decreased in patients homozygous for UGT1A1*6 or UGT1A1*28, or double heterozygotes (*6/*28). However, the reference dose for patients with these genetic polymorphisms is unclear.
Methods
We investigated the relationship between the SN-38G/SN-38 concentration ratio and the dose of CPT-11 in 70 patients with colorectal cancer who received FOLFIRI-based regimens, by measuring the plasma concentrations of CPT-11, SN-38, and SN-38G.
Results
The SN-38G/SN-38 concentration ratio was lower in patients who were homozygous for UGT1A1*6, heterozygous for UGT1A1*6 or UGT1A1*28, or were double heterozygotes compared with patients with wild-type genes. The relative decreases in the SN-38G/SN-38 concentration ratio in patients homozygous for UGT1A1*6 and in double heterozygotes were greater than in patients heterozygous for UGT1A1*6 or UGT1A1*28. Interestingly, decreases in the SN-38G/SN-38 concentration ratio were associated with decreases in the neutrophil count and the final infusion dose of CPT-11.
Conclusion
Our results suggest that the SN-38G/SN-38 concentration ratio is an important factor for guiding dose adjustments, even in patients with wild-type genes. Therefore, the SN-38G/SN-38 concentration ratio, as an index of the patient’s metabolic capacity, is useful for assessing dose adjustments of CPT-11.
In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein ...kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.
Myosin II is an essential component of the actomyosin contractile ring and plays a crucial role in cytokinesis by generating the forces necessary for contraction of the actomyosin ring. Cdc4 is an ...essential myosin II light chain in fission yeast and is required for cytokinesis. In various eukaryotes, the phosphorylation of myosin is well documented as a primary means of activating myosin II, but little is known about the regulatory mechanisms of Cdc4. Here, we isolated Nrd1, an RNA-binding protein with RNA-recognition motifs, as a multicopy suppressor of cdc4 mutants. Notably, we demonstrated that Nrd1 binds and stabilizes Cdc4 mRNA, thereby suppressing the cytokinesis defects of the cdc4 mutants. Importantly, Pmk1 mitogen-activated protein kinase (MAPK) directly phosphorylates Nrd1, thereby negatively regulating the binding activity of Nrd1 to Cdc4 mRNA. Consistently, the inactivation of Pmk1 MAPK signaling, as well as Nrd1 overexpression, stabilized the Cdc4 mRNA level, thereby suppressing the cytokinesis defects associated with the cdc4 mutants. In addition, we demonstrated the cell cycle-dependent regulation of Pmk1/Nrd1 signaling. Together, our results indicate that Nrd1 plays a role in the regulation of Cdc4 mRNA stability; moreover, our study is the first to demonstrate the posttranscriptional regulation of myosin expression by MAPK signaling.
Abstract Idiopathic carpal tunnel syndrome is the most common entrapment neuropathy in hand surgery, and it is characterized by Noninflammatory fibrosis of subsynovial connective tissues. The ...prevalence and incidence differ between male and female individuals, and the mechanism underlying this difference remains largely unclear. In the present study, we collected subsynovial connective tissues from six male and six female patients diagnosed with idiopathic carpal tunnel syndrome during surgery. We performed a comprehensive gene expression analysis using RNA sequencing to compare the gene expression profiles between male and female patients with idiopathic carpal tunnel syndrome. We identified 26 genes with significantly different expressions between male and female patients, in which POSTN , COL1A1 , and COL3A1 , which are involved in extracellular matrix organization, and IGF1 , an important fibrotic factor, were significantly upregulated in male patients. Immunohistochemistry confirmed the expression of proteins encoded by these genes in tissues, and male patients tended to show increased POSTN expression. Our results indicate that fibrosis of subsynovial connective tissues is induced by different mechanisms in male and female patients, and genes involved in extracellular matrix organization, especially POSTN , might be important factors in male patients. This study provides insight into the pathogenesis of idiopathic carpal syndrome and might contribute to the development of new treatment strategies.
The mitogen-activated protein kinase (MAPK) pathways are signal transduction mechanisms that regulate many cellular processes in eukaryotic organisms, from yeasts to mammals. Multiple MAPKs regulate ...eukaryotic gene expression in response to various extracellular stimuli through phosphorylation of transcription factors. We have been studying the Pmk1 MAPK, a homologue of the mammalian ERK/MAPK in fission yeast. The Pmk1 MAPK regulates cell integrity and cell morphology. We have previously demonstrated that Atf1, a transcription factor downstream of the stress-activated MAPK pathway, serves also as a target of the Pmk1 MAPK signaling in fission yeast. Here, we identified ecm33+ gene, encoding a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. The gene expression of ecm33+ is regulated by two transcription factors Atf1 and Mbx1. We also developed an in vivo real-time monitoring system of Atf1 or Mbx1 transcriptional activity, which enables to monitor the activation of the Pmk1 MAPK pathway by various stimuli. Finally, we demonstrated that Ecm33 is involved in the negative regulation of the Pmk1 MAPK signaling through the control of Ca2+ homeostasis. The ecm33 deleted cells displayed Ca2+ sensitivity and increased phosphorylation levels of Pmk1 MAPK. In addition, the Ecm33 overproducing cells displayed phenotypes closely similar to those of the pmk1 knockout cell. Collectively, Ecm33 plays a role in the negative feedback regulation of Pmk1 cell integrity signaling.
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