Increased di-(2-ethylhexyl) phthalate exposure poses a differential risk for adult asthma clusters.

BACKGROUND: DEHP, a common plasticizer known for its hormone-disrupting properties, has been associated with asthma. However, a significant proportion of adult asthma cases are "non-atopic", lacking a clear etiology. METHODS: In a case-control study conducted between 2011 and 2015, 365 individuals with current asthma and 235 healthy controls from Kaohsiung City were enrolled. The control group comprised individuals without asthma, Type 2 Diabetes Mellitus (T2DM), hypertension, or other respiratory/allergic conditions. The study leveraged asthma clusters (Clusters A to F) established in a prior investigation. Analysis involved the examination of urinary DEHP metabolites (MEHP and MEHHP), along with the assessment of oxidative stress, sphingolipid metabolites, and inflammatory biomarkers. Statistical analyses encompassed Spearman's rank correlation coefficients, multiple logistic regression, and multinomial logistic regression. RESULTS: Asthma clusters (E, D, C, F, A) exhibited significantly higher ORs of MEHHP exposures compared to the control group. When considering asthma-related comorbidities (T2DM, hypertension, or both), patients without comorbidities demonstrated significantly higher ORs of the sum of primary and secondary metabolites (MEHP + MEHHP) and MEHHP compared to those with asthma comorbidities. A consistent positive correlation between urinary HEL and DEHP metabolites was observed, but a consistent negative correlation between DEHP metabolites and selected cytokines was identified. CONCLUSION: The current study reveals a heightened risk of MEHHP and MEHP + MEHHP exposure in specific asthma subgroups, emphasizing its complex relationship with asthma. The observed negative correlation with cytokines suggests a new avenue for research, warranting robust evidence from epidemiological and animal studies.

Efficacy and Safety of Anti-HER2 Targeted Therapy for Metastatic HR-Positive and HER2-Positive Breast Cancer: A Bayesian Network Meta-Analysis.

Despite the development of HER2-targeted drugs, achieving favorable outcomes for patients with HR+/HER2+MBC remains challenging. This study utilized Bayesian Network Meta-analysis to compare the efficacy and safety of anti-HER2 combination regimens. The primary analysis focused on progression-free survival (PFS), while secondary analyses included objective response rate, overall survival (OS) and the incidence rate of grade 3/4 adverse events (AEs). A comprehensive search across seven databases identified 25 randomized controlled trials for inclusion in this meta-analysis. For patients eligible for endocrinotherapy, our findings revealed that dual-target combined endocrine therapy, such as Her2-mAb+Her2-mAb+Endo (HR = 0.38; 95%CrI: 0.16-0.88) and Her2-mAb+Her2-tki+Endo (HR = 0.45; 95%CrI: 0.23-0.89), significantly improved PFS compared to endocrine therapy alone. According to the surface under the cumulative ranking curves (SUCRAs), Her2-mAb+Her2-mAb+Endo and Her2-mAb+Her2-tki+Endo ranked highest in terms of PFS and OS, respectively. For patients unsuitable for endocrine therapy, anti-HER2 dual-target combined chemotherapy, such as Her2-mAb+Her2-mAb+Chem (HR = 0.76; 95%CrI: 0.6-0.96) and Her2-mAb+Her2-tki+Chem (HR = 0.48; 95%CrI: 0.29-0.81), demonstrated significant improvements in PFS compared to Her2-mAb+Chem. The results were the same when compared with Her2-tki+Chem. According to the SUCRAs, Her2-mAb+Her2-tki+Chem and Her2-mAb+Her2-mAb+Chem ranked highest for PFS and OS, respectively. Subgroup analyses consistently supported these overall findings, indicating that dual-target therapy was the optimal approach irrespective of treatment line.

Phospholipid remodeling and its derivatives are associated with COVID-19 severity.

BACKGROUND: Timely medical intervention in severe cases of coronavirus disease 2019 (COVID-19) and better understanding of the disease's pathogenesis are essential for reducing mortality, but early classification of severe cases and its progression is challenging. OBJECTIVE: We investigated the levels of circulating phospholipid metabolites and their relationship with COVID-19 severity, as well as the potential role of phospholipids in disease progression. METHODS: We performed nontargeted lipidomic analysis of plasma samples (n = 150) collected from COVID-19 patients (n = 46) with 3 levels of disease severity, healthy individuals, and subjects with metabolic disease. RESULTS: Phospholipid metabolism was significantly altered in COVID-19 patients. Results of a panel of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) and of phosphatidylethanolamine and lysophosphatidylethanolamine (LPE) ratios were significantly correlated with COVID-19 severity, in which 16 phospholipid ratios were shown to distinguish between patients with severe disease, mild disease, and healthy controls, 9 of which were at variance with those in subjects with metabolic disease. In particular, relatively lower ratios of circulating (PC16:1/22:6)/LPC 16:1 and (PE18:1/22:6)/LPE 18:1 were the most indicative of severe COVID-19. The elevation of levels of LPC 16:1 and LPE 18:1 contributed to the changes of related lipid ratios. An exploratory functional study of LPC 16:1 and LPE 18:1 demonstrated their ability in causing membrane perturbation, increased intracellular calcium, cytokines, and apoptosis in cellular models. CONCLUSION: Significant Lands cycle remodeling is present in patients with severe COVID-19, suggesting a potential utility of selective phospholipids with functional consequences in evaluating COVID-19's severity and pathogenesis.

Environmental risks and sphingolipid signatures in adult asthma and its phenotypic clusters: a multicentre study.

BACKGROUND: Adult asthma is phenotypically heterogeneous with unclear aetiology. We aimed to evaluate the potential contribution of environmental exposure and its ensuing response to asthma and its heterogeneity. METHODS: Environmental risk was evaluated by assessing the records of National Health Insurance Research Database (NHIRD) and residence-based air pollution (particulate matter with diameter less than 2.5 micrometers (PM2.5) and PM2.5-bound polycyclic aromatic hydrocarbons (PAHs)), integrating biomonitoring analysis of environmental pollutants, inflammatory markers and sphingolipid metabolites in case-control populations with mass spectrometry and ELISA. Phenotypic clustering was evaluated by t-distributed stochastic neighbor embedding (t-SNE) integrating 18 clinical and demographic variables. FINDINGS: In the NHIRD dataset, modest increase in the relative risk with time-lag effect for emergency (N=209 837) and outpatient visits (N=638 538) was observed with increasing levels of PM2.5 and PAHs. Biomonitoring analysis revealed a panel of metals and organic pollutants, particularly metal Ni and PAH, posing a significant risk for current asthma (ORs=1.28-3.48) and its severity, correlating with the level of oxidative stress markers, notably Nε-(hexanoyl)-lysine (r=0.108-0.311, p<0.05), but not with the accumulated levels of PM2.5 exposure. Further, levels of circulating sphingosine-1-phosphate and ceramide-1-phosphate were found to discriminate asthma (p<0.001 and p<0.05, respectively), correlating with the levels of PAH (r=0.196, p<0.01) and metal exposure (r=0.202-0.323, p<0.05), respectively, and both correlating with circulating inflammatory markers (r=0.186-0.427, p<0.01). Analysis of six phenotypic clusters and those cases with comorbid type 2 diabetes mellitus (T2DM) revealed cluster-selective environmental risks and biosignatures. INTERPRETATION: These results suggest the potential contribution of environmental factors from multiple sources, their ensuing oxidative stress and sphingolipid remodeling to adult asthma and its phenotypic heterogeneity.

Dimethyl sulfoxide stimulates the AhR-Jdp2 axis to control ROS accumulation in mouse embryonic fibroblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-binding protein that responds to environmental aromatic hydrocarbons and stimulates the transcription of downstream phase I enzyme-related genes by binding the cis element of dioxin-responsive elements (DREs)/xenobiotic-responsive elements. Dimethyl sulfoxide (DMSO) is a well-known organic solvent that is often used to dissolve phase I reagents in toxicology and oxidative stress research experiments. In the current study, we discovered that 0.1% DMSO significantly induced the activation of the AhR promoter via DREs and produced reactive oxygen species, which induced apoptosis in mouse embryonic fibroblasts (MEFs). Moreover, Jun dimerization protein 2 (Jdp2) was found to be required for activation of the AhR promoter in response to DMSO. Coimmunoprecipitation and chromatin immunoprecipitation studies demonstrated that the phase I-dependent transcription factors, AhR and the AhR nuclear translocator, and phase II-dependent transcription factors such as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) integrated into DRE sites together with Jdp2 to form an activation complex to increase AhR promoter activity in response to DMSO in MEFs. Our findings provide evidence for the functional role of Jdp2 in controlling the AhR gene via Nrf2 and provide insights into how Jdp2 contributes to the regulation of ROS production and the cell spreading and apoptosis produced by the ligand DMSO in MEFs.

Successful treatment of hypervirulent Klebsiella pneumoniae bacteremia with combination carbapenem and rifampicin.

Hypervirulent Klebsiella pneumoniae (hvKP) with a high mucus phenotype, can cause liver abscess and extrahepatic invasive infection. The morbidity of hvKP infections has increased recently. Here we describe a case report of septicemia caused by hvKP due to the term septic arthritis of right knee joint in a 29-year-old male. The patient was persistent fever with a peak temperature at 40.6 °C. However, based on the drug sensitivity, the treatment failed frequently. The patient did not improve clinically on susceptible monotherapy antimicrobial. Combination therapy with meropenem and rifampicin (RFP) lead to clinical improvement and discharge.

Particulate matter of 2.5 µm or less in diameter disturbs the balance of TH17/regulatory T cells by targeting glutamate oxaloacetate transaminase 1 and hypoxia-inducible factor 1α in an asthma model.

BACKGROUND: Epidemiologic evidence suggests that exposure to particulate matter of 2.5 µm or less in diameter (PM2.5) aggravates asthma. OBJECTIVE: We sought to investigate the underlying mechanisms between PM2.5 exposure and asthma severity. METHODS: The relationship between PM2.5 exposure and asthma severity was investigated in an asthma model with CD4+ T cell-specific aryl hydrocarbon receptor (AhR)-null mice. Effects of PM2.5 and polycyclic aromatic hydrocarbons (PAHs) on differentiation of TH17/regulatory T (Treg) cells were investigated by using flow cytometry and quantitative RT-PCR. Mechanisms were investigated by using mRNA sequencing, chromatin immunoprecipitation, bisulfite sequencing, and glycolysis rates. RESULTS: PM2.5 impaired differentiation of Treg cells, promoted differentiation of TH17 cells, and aggravated asthma in an AhR-dependent manner. PM2.5 and one of its prominent PAHs, indeno[1,2,3-cd]pyrene (IP), promoted differentiation of TH17 cells by upregulating hypoxia-inducible factor 1α expression and enhancing glycolysis through AhRs. Exposure to PM2.5 and IP enhanced glutamate oxaloacetate transaminase 1 (Got1) expression through AhRs and accumulation of 2-hydroxyglutarate, which inhibited ten-eleven translocation methylcytosine dioxygenase 2 activity, resulting in hypermethylation in the forkhead box P3 locus and impaired differentiation of Treg cells. A GOT1 inhibitor, (aminooxy)acetic acid, ameliorated asthma by shifting differentiation of TH17 cells to Treg cells. Similar regulatory effects of exposure to PM2.5 or IP on TH17/Treg cell imbalance were noted in human T cells, and in a case-control design PAH exposure appeared to be a potential risk factor for asthma. CONCLUSIONS: The AhR-hypoxia-inducible factor 1α and AhR-GOT1 molecular pathways mediate pulmonary responses on exposure to PM2.5 through their ability to disturb the balance of TH17/Treg cells.

Aryl hydrocarbon receptor signaling promotes ORMDL3-dependent generation of sphingosine-1-phosphate by inhibiting sphingosine-1-phosphate lyase.

Aryl hydrocarbon receptor (AhR), a cellular chemical sensor, controls cellular homeostasis, and sphingosine-1-phosphate (S1P), a bioactive intermediate of sphingolipid metabolism, is believed to have a role in immunity and inflammation, but their potential crosstalk is currently unknown. We aimed to determine whether there is a functional linkage between AhR signaling and sphingolipid metabolism. We showed that AhR ligands, including an environmental polycyclic aromatic hydrocarbon (PAH), induced S1P generation, and inhibited S1P lyase (S1PL) activity in resting cells, antigen/IgE-activated mast cells, and mouse lungs exposed to the AhR ligand alone or in combination with antigen challenge. The reduction of S1PL activity was due to AhR-mediated oxidation of S1PL at residue 317, which was reversible by the addition of an antioxidant or in cells with knockdown of the ORMDL3 gene encoding an ER transmembrane protein, whereas C317A S1PL mutant-transfected cells were resistant to the AhR-mediated effect. Furthermore, analysis of AhR ligand-treated cells showed a time-dependent increase of the ORMDL3-S1PL complex, which was confirmed by FRET analysis. This change increased the S1P levels, which in turn, induced mast cell degranulation via S1PR2 signaling. In addition, elevated levels of plasma S1P were found in children with asthma compared to non-asthmatic subjects. These results suggest a new regulatory pathway whereby the AhR-ligand axis induces ORMDL3-dependent S1P generation by inhibiting S1PL, which may contribute to the expression of allergic diseases.

Correction to: Aryl hydrocarbon receptor signaling promotes ORMDL3-dependent generation of sphingosine-1-phosphate by inhibiting sphingosine-1-phosphate lyase.

In this article, published online 23 March 2018, the affiliation 10 of Zhou Y was incorrect. The affiliation should be "Children's Hospital and Institute of Biomedical Sciences, Fudan University. Key Laboratory of Neonatal Disease, Ministry of Health, 201102, Shanghai, China." The authors regret the errors.

Restoration of hydroxyindole O-methyltransferase levels in human cancer cells induces a tryptophan-metabolic switch and attenuates cancer progression.

5-Methoxytryptophan (5-MTP) is a tryptophan metabolite with recently discovered anti-inflammatory and tumor-suppressing activities. Its synthesis is catalyzed by a hydroxyindole O-methyltransferase (HIOMT)-like enzyme. However, the exact identity of this HIOMT in human cells remains unclear. Human HIOMT exists in several alternatively spliced isoforms, and we hypothesized that 5-MTP-producing HIOMT is a distinct isoform. Here, we show that human fibroblasts and cancer cells express the HIOMT298 isoform as contrasted with the expression of the HIOMT345 isoform in pineal cells. Sequencing analysis of the cloned isoforms revealed that HIOMT298 is identical to the sequence of a previously reported truncated HIOMT isoform. Of note, HIOMT298 expression was reduced in cancer cells and tissues. Stable transfection of A549 cancer cells with HIOMT298 restored HIOMT expression to normal levels, accompanied by 5-MTP production. Furthermore, HIOMT298 transfection caused a tryptophan-metabolic switch from serotonin to 5-MTP production. To determine the in vivo relevance of this alteration, we compared growth and lung metastasis of HIOMT298-transfected A549 cells with those of vector- or untransfected A549 cells as controls in a murine xenograft model. Of note, the HIOMT298-transfected A549 cells exhibited slower growth and lower metastasis than the controls. Our findings provide insight into the crucial role of HIOMT298 in 5-MTP production in cells and in inhibiting cancer progression and highlight the potential therapeutic value of 5-MTP for managing cancer.

A prominent air pollutant, Indeno[1,2,3-cd]pyrene, enhances allergic lung inflammation via aryl hydrocarbon receptor.

Chronic exposure to ambient polycyclic aromatic hydrocarbons (PAHs) is associated with asthma, but its regulatory mechanisms remain incompletely defined. We report herein that elevated levels of urinary 1-hydroxypyrene, a biomarker of PAH exposure, were found in asthmatic subjects (n = 39) as compared to those in healthy subjects (n = 43) living in an industrial city of Taiwan, where indeno[1,2,3-cd]pyrene (IP) was found to be a prominent PAH associated with ambient PM2.5. In a mouse model, intranasal exposure of mice with varying doses of IP significantly enhanced antigen-induced allergic inflammation, including increased airway eosinophilia, Th2 cytokines, including IL-4 and IL-5, as well as antigen-specific IgE level, which was absent in dendritic cell (DC)-specific aryl hydrocarbon receptor (AhR)-null mice. Mechanistically, IP treatment significantly altered DC's function, including increased level of pro-inflammatory IL-6 and decreased generation of anti-inflammatory IL-10. The IP's effect was lost in DCs from mice carrying an AhR-mutant allele. Taken together, these results suggest that chronic exposure to environmental PAHs may pose a significant risk for asthma, in which IP, a prominent ambient PAH in Taiwan, was shown to enhance the severity of allergic lung inflammation in mice through, at least in part, its ability in modulating DC's function in an AhR-dependent manner.

Inhibition of cancer cell epithelial mesenchymal transition by normal fibroblasts via production of 5-methoxytryptophan.

We reported previously that human fibroblasts release 5-methoxytryptophan (5-MTP) which inhibits cancer cell COX-2 overexpression and suppresses cancer cell migration and metastasis. To determine whether fibroblasts block cancer cell epithelial mesenchymal transition (EMT) via 5-MTP, we evaluated the effect of Hs68 fibroblasts (HsFb) on A549 cancer cell EMT in a two-chamber system. Co-incubation of A549 with HsFb prevented TGF-ß1-induced reduction of E-cadherin and increase in Snail and N-cadherin. Transfection of HsFb with tryptophan hydroxylase-1 siRNA, which inhibited tryptophan hydroxylase-1 protein expression and 5-MTP release in HsFb abrogated the effect of HsFb on A549 EMT. Direct addition of pure 5-MTP to cultured A549 cells followed by TGF-ß1 prevented TGF-ß1-induced reduction of E-cadherin, and elevation of Snail, vimentin and matrix metalloproteinase 9. Administration of 5-MTP to a murine xenograft tumor model reduced vimentin protein expression in the tumor tissues compared to vehicle control which was correlated with reduction of metastasis in the 5-MTP treated mice. Our experimental data suggest that 5-MTP exerted its anti-EMT actions through inhibition of p38 MAPK activation, p65/p50 NF-κB nuclear translocation and transactivation without the involvement of COX-2 or p300 histone acetyltransferase. Our findings indicate that fibroblasts release a tryptophan metabolite, 5-MTP, to reduce cancer cell EMT, migration, invasion and metastasis.

Prostacyclin and PPARα agonists control vascular smooth muscle cell apoptosis and phenotypic switch through distinct 14-3-3 isoforms.

We hypothesized that prostacyclin (PGI2) protects vascular smooth muscle cell (VSMC) against apoptosis and phenotypic switch through peroxisome proliferator-activated receptor-α (PPARα) activation and 14-3-3 upregulation. Here we showed that transfection of rat aortic VSMC, A-10, with PGI2-producing vectors, Ad-COPI, resulted in attenuated H2O2-induced apoptosis accompanied by a selective increase in 14-3-3ß and 14-3-3θ expression. Carbaprostacyclin (cPGI2) and Wy14,643 exerted a similar effect. The effects of PGI2 were abrogated by MK886, a PPARα antagonist, but not GSK3787, a PPARδ antagonist. PPARα transfection upregulated 14-3-3ß and θ expression and attenuated H2O2-induced apoptosis. H2O2-induced 14-3-3ß but not 14-3-3θ degradation was blocked by a caspase 3 inhibitor. Furthermore, 14-3-3ß but not 14-3-3θ overexpression reduced, while 14-3-3ß siRNA aggravated apoptosis. VSMC contractile proteins and serum response factor (SRF) were reduced in H2O2-treated A-10 cells which were concurrently prevented by caspase 3 inhibitor. By contrast, PGI2 prevented H2O2-induced SM22α and Calponin-1 degradation without influencing SRF. cPGI2 and Wy14,643 also effectively blocked VSMC phenotypic switch induced by growth factors (GFs). GFs suppressed 14-3-3ß, θ, ε and η isoforms and cPGI2 prevented the decline of ß, θ and η, but not ε. 14-3-3θ siRNA abrogated the protective effect of cPGI2 on SM22α and Calponin-1 while 14-3-3 θ or 14-3-3ß overexpression partially restored SM22α. These results indicated that PGI2 protects VSMCs via PPARα by upregulating 14-3-3ß and 14-3-3θ. 14-3-3ß upregulation confers resistance to apoptosis whereas 14-3-3θ and ß upregulation protects SM22α and Calponin-1 from degradation.

Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan.

Cyclooxygenase-2 (COX-2) expression is induced by mitogenic and proinflammatory factors. Its overexpression plays a causal role in inflammation and tumorigenesis. COX-2 expression is tightly regulated, but the mechanisms are largely unclear. Here we show the control of COX-2 expression by an endogenous tryptophan metabolite, 5-methoxytryptophan (5-MTP). By using comparative metabolomic analysis and enzyme-immunoassay, our results reveal that normal fibroblasts produce and release 5-MTP into the extracellular milieu whereas A549 and other cancer cells were defective in 5-MTP production. 5-MTP was synthesized from L-tryptophan via tryptophan hydroxylase-1 and hydroxyindole O-methyltransferase. 5-MTP blocked cancer cell COX-2 overexpression and suppressed A549 migration and invasion. Furthermore, i.p. infusion of 5-MTP reduced tumor growth and cancer metastasis in a murine xenograft tumor model. We conclude that 5-MTP synthesis represents a mechanism for endogenous control of COX-2 overexpression and is a valuable lead for new anti-cancer and anti-inflammatory drug development.

Nestin is essential for zebrafish brain and eye development through control of progenitor cell apoptosis.

BACKGROUND: Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear. METHODOLOGY/PRINCIPAL FINDINGS: As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO. CONCLUSION/SIGNIFICANCE: These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis.

Developmental gene regulatory networks in the zebrafish embryo.

The genomic developmental program operates mainly through the regulated expression of genes encoding transcription factors and signaling pathways. Complex networks of regulatory genetic interactions control developmental cell specification and fates. Development in the zebrafish, Danio rerio, has been studied extensively and large amounts of experimental data, including information on spatial and temporal gene expression patterns, are available. A wide variety of maternal and zygotic regulatory factors and signaling pathways have been discovered in zebrafish, and these provide a useful starting point for reconstructing the gene regulatory networks (GRNs) underlying development. In this review, we describe in detail the genetic regulatory subcircuits responsible for dorsoanterior-ventroposterior patterning and endoderm formation. We describe a number of regulatory motifs, which appear to act as the functional building blocks of the GRNs. Different positive feedback loops drive the ventral and dorsal specification processes. Mutual exclusivity in dorsal-ventral polarity in zebrafish is governed by intra-cellular cross-inhibiting GRN motifs, including vent/dharma and tll1/chordin. The dorsal-ventral axis seems to be determined by competition between two maternally driven positive-feedback loops (one operating on Dharma, the other on Bmp). This is the first systematic approach aimed at developing an integrated model of the GRNs underlying zebrafish development. Comparison of GRNs' organizational motifs between different species will provide insights into developmental specification and its evolution. The online version of the zebrafish GRNs can be found at http://www.zebrafishGRNs.org.

Overexpression of an Aurora-C kinase-deficient mutant disrupts the Aurora-B/INCENP complex and induces polyploidy.

Aurora kinases are emerging as key regulators of centrosome function, chromosome segregation and cytokinesis. We previously isolated Aurora-C (Aie1), a third type of Aurora kinase, in a screen for kinases expressed in mouse sperm and eggs. Currently, we know very little about the precise localization and function of Aurora-C. Immunofluorescence analysis of ectopically expressed GFP-Aurora-C has revealed that Aurora-C is a new member of the chromosomal passenger proteins localizing first to the centromeres and then to the central spindles during cytokinesis. In order to study the potential role of Aurora-C, we examined the effects of a kinase-deficient (KD) mutant (AurC-KD) in HeLa Tet-Off cells under tetracycline control. Our results showed that overexpression of AurC-KD causes defects in cell division and induces polyploidy and apoptosis. Interestingly, AurC-KD overexpression also inhibits centromere/kinetochore localization of Aurora-B, Bub1, and BubR1, reduces histone H3 phosphorylation, and disrupts the association of INCENP with Aurora-B. Together, our results showed that Aurora-C is a chromosomal passenger protein, which may serve as a key regulator in cell division.

Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly.

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.