Precursor T lymphoblastic lymphoma (T-LBL) often manifests as a mediastinal mass sometimes compressing vital structures like vessels or large airways. This case was a 40-year-old male who developed T-LBL presenting as respiratory failure caused by mediastinal T-LBL. He presented with persistent life threatening hypoxia despite tracheal intubation. We successfully managed this respiratory failure using venovenous (VV) ECMO. Induction chemotherapy was started after stabilizing oxygenation and the mediastinal lesion shrank rapidly. Respiratory failure caused by compression of the central airway by tumor is an oncologic emergency. VV ECMO may be an effective way to manage this type of respiratory failure as a bridge to chemotherapy.
Synthetic lethal interactions enable a novel approach for discovering specific genetic vulnerabilities in cancer cells that can be exploited for the development of therapeutics. Despite successes in model organisms such as yeast, discovering synthetic lethal interactions on a large scale in human cells remains a significant challenge. We describe a comparative genomic strategy for identifying cancer relevant synthetic lethal interactions whereby candidate interactions are prioritized based on genetic interaction data available in yeast, followed by targeted testing of candidate interactions in human cell lines. As proof of principle, we describe two novel synthetic lethal interactions in human cells discovered by this approach, one between the tumor suppressor gene SMARCB1 and PSMA4, and another between alveolar soft-part sarcoma-associated ASPSCR1 and PSMC2. These results suggest therapeutic targets for cancers harboring mutations in SMARCB1 or ASPSCR1, and highlight the potential of a targeted, cross-species strategy for identifying synthetic lethal interactions relevant to human cancer.
Aberrant protein aggregation is a dominant pathological feature in neurodegenerative diseases. Protein aggregates cannot be processed by the proteasome; instead, they are frequently concentrated to a perinuclear inclusion body, the aggresome, and subsequently removed by autophagy. Paradoxically, proteasomes are also concentrated at aggresomes and other related inclusion bodies prevalent in neurodegenerative disease. Here, we show that proteasomes are crucial components in aggresome clearance. The disassembly and disposal of aggresomes requires a proteasomal deubiquitinating enzyme, Poh1, which cleaves ubiquitinated proteins and releases ubiquitin chains. In Poh1-deficient cells, aggresome clearance is blocked. Remarkably, microinjection of free lysine (K) 63-linked ubiquitin chains restores aggresome degradation. We present evidence that free ubiquitin chains produced by Poh1 bind and activate the deacetylase, HDAC6, which in turn stimulates actinomyosin- and autophagy-dependent aggresome processing. Thus, unanchored ubiquitin chains are key signaling molecules that connect and coordinate the proteasome and autophagy to eliminate toxic protein aggregates.
Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3′-end down-regulation. Removal of SSA from the culture media restored expression of the 3′ ends of genes, suggesting that U2 snRNP is required for expression of the 3′ end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5′ end of 3′-end down regulated genes, such as CDK6, SMEK2 and EGFR, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation.
Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions.
We present a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds. We developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes. The MoBY-ORF resource has numerous genetic and chemical-genetic applications, but here we focus on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays. The complementation cloning was validated by mutation detection using whole-genome yeast tiling microarrays, which identified unique polymorphisms associated with a drug-resistant mutant. We used the MoBY-ORF library to identify the genetic basis of several drug-resistant mutants and in this analysis discovered a new class of sterol-binding compounds.
The fission yeast Schizosaccharomyces pombe has more metazoan-like features than the budding yeast Saccharomyces cerevisiae, yet it has similarly facile genetics. Here, we present a large-scale verified binary protein-protein interactome network, “StressNet”, based on high-throughput yeast two-hybrid screens of interacting proteins classified as part of stress-response and signal transduction pathways in S. pombe. We performed systematic, cross-species interactome mapping using StressNet and a protein interactome network of orthologous proteins in S. cerevisiae. With cross-species comparative network studies, we detected a previously unidentified component (Snr1) of the S. pombe mitogen-activated protein kinase Sty1 pathway. Coimmunoprecipitation experiments showed that Snr1 interacted with Sty1 and that deletion of snr1 increased the sensitivity of S. pombe cells to stress. Comparison of StressNet with the interactome network of orthologous proteins in S. cerevisiae showed that the majority of interactions among these stress-response and signaling proteins are not conserved between species, but are “rewired;” orthologous proteins have different binding partners in both species. In particular, transient interactions connecting proteins in different functional modules were more likely to be rewired than conserved. By directly testing interactions between proteins in one yeast species and their corresponding binding partners in the other yeast species with yeast two-hybrid assays, we found that about half of the interactions traditionally considered “conserved” form modified interaction interfaces that may potentially accommodate novel functions.
Histone acetyltransferase enzymes (HATs) are important therapeutic targets, but there are few cell-based assays available for evaluating the pharmacodynamics of HAT inhibitors. Here we present the application of a FRET-based reporter, Histac, in live cell studies of p300/CBP HAT inhibition, by both genetic and pharmacologic disruption. shRNA knockdown of p300/CBP led to increased Histac FRET, suggesting a role for p300/CBP in the acetylation of the histone H4 tail present. Additionally, we describe a new p300/CBP HAT inhibitor, C107, and show that it can also increase cellular Histac FRET. Taken together, these studies provide a live cell strategy for identifying and evaluating p300/CBP inhibitors.
drug design; enzymes; FRET; protein modifications; Histone H4
An open-label, prospective, multicenter study was conducted between October 2006 and March 2010 to assess the efficacy and safety of intravenous voriconazole (VRCZ) as empirical therapy for antibiotic-refractory febrile neutropenia in Japanese patients with hematological disorders. In addition, to find the patient groups that may benefit from antifungal therapy, the definition of invasive fungal infection proposed by EORTC/MSG (2002) was assessed in this study. Plasma (1-3)-β-d-glucan and Aspergillus PCR in blood were also measured to improve the diagnostic accuracy. A total of 103 patients (median age, 59 years), including 25 undergoing induction chemotherapies and 19 allogeneic hematopoietic cell transplants, were evaluable. Sixty-nine percent of the patients achieved resolution of clinical symptoms and 31 % achieved treatment success, defined as fulfilling the previously described five-part composite endpoint. Although VRCZ was discontinued in 9.7 % of the patients because of adverse effects, all the patients recovered soon after discontinuation of VRCZ. The treatment success rate of VRCZ appeared to be higher in patients categorized as “not classified” compared with “possible invasive fungal disease” according to the EORTC/MSG criteria. Moreover, six “not classified” patients were positive for either plasma (1-3)-β-d-glucan (n = 5) or Aspergillus PCR in blood (n = 2). The present study demonstrates that empirical VRCZ therapy is safe and effective in Japanese patients. Additionally, (1-3)-β-d-glucan and Aspergillus PCR tests were expected to provide additional information on the diagnosis of invasive fungal infections.
Prospective multicenter study; Voriconazole; Empirical antifungal therapy
The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ− mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ− cells and increased deletion mutagenesis at the ori5 region in ρ+ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.
Two highly modified linear tetrapeptides, padanamides A (1) and B (2), are produced by laboratory cultures of a Streptomyces sp. obtained from a marine sediment. Padanamide B is cytotoxic to Jurkat cells and a chemical genomics analysis using Saccharomyces cerevisiae deletion mutants suggested that padanamide A inhibits cysteine and methionine biosynthesis or that these amino acids are involved in the yeast’s response to the peptide.
Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.
Elongator is required for the synthesis of the mcm5s2 modification found on tRNAs recognizing AA-ending codons. In order to obtain a global picture of the role of Elongator in translation, we used reverse protein arrays to screen the fission yeast proteome for translation defects. Unexpectedly, this revealed that Elongator inactivation mainly affected three specific functional groups including proteins implicated in cell division. The absence of Elongator results in a delay in mitosis onset and cytokinesis defects. We demonstrate that the kinase Cdr2, which is a central regulator of mitosis and cytokinesis, is under translational control by Elongator due to the Lysine codon usage bias of the cdr2 coding sequence. These findings uncover a mechanism by which the codon usage, coupled to tRNA modifications, fundamentally contributes to gene expression and cellular functions.
Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis.
We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.
As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins.
This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.
A combination of several rate-limiting steps allows for efficient control of alternative splicing.
Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2–GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules.
Post-translational modifications (PTMs) have a key role in regulating cell functions. Consequently, identification of PTM sites has a significant impact on understanding protein function and revealing cellular signal transductions. Especially, phosphorylation is a ubiquitous process with a large portion of proteins undergoing this modification. Experimental methods to identify phosphorylation sites are labor-intensive and of high-cost. With the exponentially growing protein sequence data, development of computational approaches to predict phosphorylation sites is highly desirable.
Here, we present a simple and effective method to recognize phosphorylation sites by combining sequence patterns and evolutionary information and by applying a novel noise-reducing algorithm. We suggested that considering long-range region surrounding a phosphorylation site is important for recognizing phosphorylation peptides. Also, from compared results to AutoMotif in 36 different kinase families, new method outperforms AutoMotif. The mean accuracy, precision, and recall of our method are 0.93, 0.67, and 0.40, respectively, whereas those of AutoMotif with a polynomial kernel are 0.91, 0.47, and 0.17, respectively. Also our method shows better or comparable performance in four main kinase groups, CDK, CK2, PKA, and PKC compared to six existing predictors.
Our method is remarkable in that it is powerful and intuitive approach without need of a sophisticated training algorithm. Moreover, our method is generally applicable to other types of PTMs.
Ran GTPase activates several target molecules to induce microtubule formation around the chromosomes and centrosomes. In fission yeast, in which the nuclear envelope does not break down during mitosis, Ran targets the centrosomal transforming acidic coiled-coil (TACC) protein Alp7 for spindle formation. Alp7 accumulates in the nucleus only during mitosis, although its underlying mechanism remains elusive. Here, we investigate the behaviour of Alp7 and its binding partner, Alp14/TOG, throughout the cell cycle. Interestingly, Alp7 enters the nucleus during interphase but is subsequently exported to the cytoplasm by the Exportin-dependent nuclear export machinery. The continuous nuclear export of Alp7 during interphase is essential for maintaining the array-like cytoplasmic microtubule structure. The mitosis-specific nuclear accumulation of Alp7 seems to be under the control of cyclin-dependent kinase (CDK). These results indicate that the spatiotemporal regulation of microtubule formation is established by the Alp7/TACC–Alp14/TOG complex through the coordinated interplay of Ran and CDK.
cyclin-dependent kinase; microtubule; nuclear transport; Ran GTPase; TACC–TOG
Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho–] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho–] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.
Although a long latency period of toxicity after exposure to methylmercury (MeHg) is known to exist in humans, few animal studies have addressed this issue. Substantiation of delayed MeHg toxicity in animals would affect the risk evaluation of MeHg.
Our goal in this study was to demonstrate the existence of a latency period in a rodent model in which the toxicity of perinatal MeHg exposure becomes apparent only later in life. Our study included metallothionein (MT) knockout mice because studies have suggested the potential susceptibility of this strain to the neurodevelopmental toxicity of MeHg.
Pregnant MT-null and wild-type C57Bl/6J mice were exposed to MeHg through their diet containing 5 μg Hg/g during gestation and early lactation. We examined behavioral functions of the offspring using frequently used paradigms, including open field behavior (OPF), passive avoidance (PA), and the Morris water maze (MM), at ages of 12–13 and 52–53 weeks.
At 12 weeks of age, behavioral effects of MeHg were not detected, except for OPF performance in MeHg-exposed MT-null females. At 52 weeks of age, the MeHg-exposed groups showed poorer performance both in PA and MM, and their OPF activity differed from controls. These effects of MeHg appeared exaggerated in the MT-null strain. The brain Hg concentration had leveled off by 13 weeks of age.
The results suggest the existence of a long latency period after perinatal exposure to low-level MeHg, in which the behavioral effects emerged long after the leveling-off of brain Hg levels. Hence, the initial toxicologic event responsible for the late effects should have occurred before this leveling-off of brain Hg.
behavioral effects; latency; methylmercury; mice; perinatal exposure
The aim of this study was to determine the level of exposure of mercury (Hg) miners and smelter workers to elemental mercury (Hg0) vapor in China, who work in Hg mines without using protective equipment against Hg0 vapor. In addition, the level of methylmercury (MeHg) intake by the workers was estimated from the MeHg concentration in their hair.
Urinary total mercury (THg) and hair THg and MeHg concentrations were measured in 26 Hg miners and smelter workers (i.e., exposed group), and 48 unexposed people (unexposed group).
The exposed group showed high geometric mean THg concentrations in urine (258 ng/ml, 226 μg/g creatinine) and hair (20.0 μg/g). The urinary THg concentration of the smelter workers in particular was extremely high (338 μg/g creatinine in urine). The highest urine THg concentration reached 4577 μg/g creatinine. THg concentrations in urine and hair showed a significant correlation in the exposed group (r=0.62), indicating the adhesion of Hg0 vapor to hair. However, no such significant correlation was found in the unexposed group. Hair MeHg concentration in the exposed group (1.97 μg/g) was about threefold higher than that in the unexposed group (0.60 μg/g).
This study shows that smelter workers in a Chinese Hg mine are exposed to extremely high levels of Hg0 vapor, and that Hg miners are exposed to higher levels of MeHg than the unexposed subjects. Further study is needed to determine the cause of the higher hair MeHg concentration in the exposed group.
mercury mine; elemental mercury; hair; urine; methylmercury
The Cdc25 phosphatase promotes entry into mitosis through the removal of inhibitory phosphorylations on the Cdc2 subunit of the Cdc2/CyclinB complex. During interphase, or after DNA damage, Cdc25 is suppressed by phosphorylation at Ser287 (Xenopus numbering; Ser216 of human Cdc25C) and subsequent binding of the small acidic protein, 14-3-3. As reported recently, at the time of mitotic entry, 14-3-3 protein is removed from Cdc25 and S287 is dephosphorylated by protein phosphatase 1 (PP1). After the initial activation of Cdc25 and consequent derepression of Cdc2/CyclinB, Cdc25 is further activated through a Cdc2-catalyzed positive feedback loop. Although the existence of such a loop has been appreciated for some time, the molecular mechanism for this activation has not been described. We report here that phosphorylation of S285 by Cdc2 greatly enhances recruitment of PP1 to Cdc25, thereby accelerating S287 dephosphorylation and mitotic entry. Moreover, we show that two other previously reported sites of Cdc2-catalyzed phosphorylation on Cdc25 are required for maximal biological activity of Cdc25, but they do not contribute to PP1 regulation and do not act solely through controlling S287 phosphorylation. Therefore, multiple mechanisms, including enhanced recruitment of PP1, are used to promote full activation of Cdc25 at the time of mitotic entry.
Several lines of evidence suggest that gene expression is regulated not only by the interaction between transcription factors and DNA but also by the higher-order architecture of the cell nucleus. PML bodies are one of the most prominent nuclear substructures which have been implicated in transcription regulation during apoptosis and stress responses. Bach2 is a member of the BTB-basic region leucine zipper factor family and represses transcription activity directed by the 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element. Bach2 forms nuclear foci associated with PML bodies upon oxidative stress. Here, we demonstrate that transcription activity associated with PML bodies is selectively repressed by the recruitment of Bach2 around PML bodies. Fluorescence recovery after photobleaching experiments revealed that Bach2 showed rapid turnover in the nuclear foci. The Bach2 N-terminal region including the BTB domain is essential for the focus formation. Sumoylation of Bach2 is required for the recruitment of the protein around PML bodies. These observations represent the first example of modulation of transcription activity associated with PML bodies by a sequence-specific transcription factor upon oxidative stress.