Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks

Post-replication DNA repair facilitates the resumption of DNA synthesis upon replication fork stalling at DNA damage sites. Despite the importance of RAD18 and polymerase η (Polη) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248–282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Polη to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18 -knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR.     

The role of ferritin in the intracellular distribution of gallium 67

The binding of gallium 67 or iron 59 to ferritin in vitro was investigated using equilibrium dialysis. Gallium 67 did not bind to apo-ferritin until the protein was transformed into ferritin in the presence of iron citrate. Apotransferrin inhibited the binding of ⁶⁷Ga to ferritin, especially in the presence of sodium bicarbonate and citrate, thus indicating that ⁶⁷Ga has not gained access to ferritin from its complex with transferrin. Similar inhibition was observed for ferritin-⁵⁹Fe. The release of ⁵⁹Fe from its transferrin complex was enhanced by ATP, citrate, or ascorbic acid, while these reagents did not stimulate the dissociation of ⁶⁷Ga from its transferrin complex. On the other hand, ⁶⁷Ga injected intravenously in vivo was not found in the ferritin fractions of rat liver, kidney, and tumor. The difference between experimental results in vivo and in vitro supports the hypothesis that ⁶⁷Ga in the cytoplasm is not labile enough to be bound to ferritin. We have indicated a significant role of ferritin in distinguishing between ⁶⁷Ga and ⁵⁹Fe in the cell, and provided some clues to interpret the chemical forms of ⁶⁷Ga in the cytoplasm.     

Loss of mTOR complex 1 induces developmental blockage in early T-lymphopoiesis and eradicates T-cell acute lymphoblastic leukemia cells

mTOR is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants. Recent studies have shown that fine-tuning of the activity of mTOR complexes contributes to organogenesis and tumorigenesis. Although rapamycin, an allosteric mTOR inhibitor, is an effective immunosuppressant, the precise roles of mTOR complexes in early T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the development of both early T-cell progenitors and leukemia. Deletion of Raptor, an essential component of mTORC1, produced defects in the earliest development of T-cell progenitors in vivo and in vitro. Deficiency of Raptor resulted in cell cycle abnormalities in early T-cell progenitors that were associated with instability of the Cyclin D2/D3-CDK6 complexes; deficiency of Rictor, an mTORC2 component, did not have the same effect, indicating that mTORC1 and -2 control T-cell development in different ways. In a model of myeloproliferative neoplasm and T-cell acute lymphoblastic leukemia (T-ALL) evoked by Kras activation, Raptor deficiency dramatically inhibited the cell cycle in oncogenic Kras-expressing T-cell progenitors, but not myeloid progenitors, and specifically prevented the development of T-ALL. Although rapamycin treatment significantly prolonged the survival of recipient mice bearing T-ALL cells, rapamycin-insensitive leukemia cells continued to propagate in vivo. In contrast, Raptor deficiency in the T-ALL model resulted in cell cycle arrest and efficient eradication of leukemia. Thus, understanding the cell-context–dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets.mTOR is a serine/threonine kinase that has a central role in the regulation of cell growth and cell metabolism and forms two functionally different complexes, named mTORC1 and mTORC2 (1). The Raptor subunit is specific to the mTORC1 complex, and Rictor is specific to mTORC2. One of the major upstream signal transduction pathways of mTORC1 is the phosphatidylinositol-3 kinase (PI3K)-AKT pathway. AKT activates mTORC1 via PRAS40 and the tuberous sclerosis 1/2 (TSC1/2)-Rheb pathway. The TSC1/2 complex is an established mTORC1 suppressor, and its protein destabilization via extracellular-signal–regulated kinase (ERK) activates mTORC1 (2). Because the GTP-bound form of Ras interacts with and activates PI3K and ERK, Ras is also an activator of mTORC1 (3).Abnormalities of mTOR signals are frequently detected in patients with one of several types of leukemia (4, 5). In particular, alterations in PTEN, PI3K, or AKT frequently occur in patients with T-cell acute lymphoblastic leukemia (T-ALL) (6). In a mouse model, deletion of Pten during hematopoiesis demonstrated that Pten is critical for suppressing the development of leukemia (7–9). Furthermore, studies using Raptor- or Rictor-deficient mice revealed that activation of mTORC1 or -2 is required for the leukemogenesis evoked by Pten loss (10, 11). However, the involvement of mTORC1 in leukemogenesis associated with other oncogenic signals, such as Ras, is not well understood. More importantly, it has remained unclear whether mTORC1 inactivation would eradicate T-ALL.Rapamycin is a potent immunosuppressant that induces severe thymic atrophy in rodents. However, a study of conditional deletion of Rheb, which encodes an mTORC1 activator, or of mTOR with a Cd4-Cre transgene showed that mTORC1 inactivation does not result in apparent thymic phenotypes under steady-state conditions (12), leading to the possibility that rapamycin may affect T-cell development in an mTORC1-independent manner. In addition, it has been reported that 4E-BP1 is a rapamycin-insensitive mTORC1 substrate, suggesting that rapamycin treatment does not necessarily represent mTORC1 inactivation (13). Thus, the precise roles of mTOR complexes in T-cell development remain unclear.In this study, we focused on the role of mTOR in T-cell development. Our data clearly show that mTORC1, but not mTORC2, is essential for cell cycling of the earliest T-cell progenitors, but not myeloid progenitors. In addition, we found that mTORC1 inactivation effectively prevented the induction of T-ALL, but not myeloproliferative neoplasm (MPN), induced by oncogenic Kras, indicating that mTORC1 is specifically essential for T-cell development and leukemogenesis. Importantly, we revealed that inactivation of mTORC1 by Raptor deficiency efficiently eradicates Notch-driven T-ALL in vivo. Thus, dissection of mTOR signals in vivo should suggest therapeutic approaches that will successfully eradicate many types of cancer.     

Defective development of the gall bladder and cystic duct in Lgr4‐ hypomorphic mice

Leucine‐rich repeat (LRR) ‐containing G protein coupled receptor (LGR) family members are characterized by the presence of a seven‐transmembrane domain and LRR motifs. We describe a new function for Lgr4 in the development of the gall bladder and cystic duct and in the epithelium–mesenchyme interaction. Lgr4 expression was observed in the gall bladder epithelium when the gall bladder primordium elongated ventrally. Although Lgr4 hypomorphic mutant (Lgr4^Gt/Gt) embryos developed a normal gall bladder bud at embryonic day (E) 10.25, no further elongation was observed at later stages. At E12.5, the mesenchyme surrounding the gall bladder had completely disappeared in Lgr4^Gt/Gt embryos, while the gall bladder remained unelongated. Neighboring tissues such as liver and pancreas were unaffected, as revealed by expression of marker genes. This is the first report of a mutant mouse that lacks a gall bladder and cystic duct without affecting the other tissues that derive from the same hepatic diverticulum. Developmental Dynamics 238:993–1000, 2009. © 2009 Wiley‐Liss, Inc.     

Valproic acid-induced neutropenia

OBJECTIVE: To report a case of severe neutropenia caused by valproic acid (VPA). CASE SUMMARY: A 56-year-old white woman with an infectious brain abscess causing tonic-clonic seizure activity was treated with VPA. She developed severe neutropenia after 2 days of VPA therapy. The absolute neutrophil count reached a nadir of 47 cells/mm(3) during VPA use and returned to normal upon its discontinuation. DISCUSSION: VPA is considered to be a well-tolerated antiepileptic drug. While neutropenia has been reported, it has been mild and transient. This patient developed severe neutropenia during effective treatment with VPA, making her significantly susceptible to infection. The Naranjo probability scale indicates VPA as the probable cause of neutropenia in this case. CONCLUSIONS: This report of severe neutropenia caused by VPA emphasizes the importance of monitoring complete blood cell counts during therapy with this agent.     

Can cognitive leisure activity prevent cognitive decline in older adults? A systematic review of intervention studies

The aims of this systematic review were to investigate what kind of cognitive leisure activities have been used in intervention studies targeting older adults, and whether these activities improve cognitive function or inhibit cognitive decline. Based on the PRISMA declaration, we searched keywords using three electronic databases: PubMed, PsycINFO and PsycARTICLES. Intervention studies involving cognitive leisure activities with cognitive assessments set as outcomes were included. We regarded cognitive leisure activities as activities for enjoyment or well‐being that cause intellectual stimulation (e.g. reading, playing board games). To investigate the influence of each activity on cognitive domains, multicomponent programs (e.g. combined music and art) were excluded. In total, 20 studies were included in the evaluation. Consequently, intervention studies related to arts, writing, board games, reading, handicrafts, a crossword puzzle and learning computer skills were identified. Of the 20 studies, 13 showed improvement in some cognitive domain. In 12 of these 13 studies, the intervention effects were not observed in a specific cognitive domain; rather, the intervention effects were observed across multiple cognitive domains and on working memory. The results of the present review suggest that cognitive function in older adults can be improved through cognitive leisure activity interventions. Activities related to learning new skills, that cause strong intellectual stimulation and that include communication elements were considered particularly effective tools. However, as the number of studies is small, more high‐quality research needs to be accumulated. Geriatr Gerontol Int 2019; 19: 469–482 .     

Production of antitumor substances from shochu distillation remnants

This study aimed to develop a new processing method for the effective use of rice shochu distillation remnants. We examined the inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with various fungi. Interestingly, high inhibitory effects on the growth of human lung carcinoma cells in the medium of rice shochu distillation remnants with Aspergillus oryzae were obtained, although no inhibitory effect was observed in the case of synthetic medium. We therefore fractionated the medium of rice shochu distillation remnants with A. oryzae using anion-exchange and reverse-phase chromatography. Furthermore, we attempted to determine the chemical structure of compounds that showed high inhibitory effects on the growth of tumor cells. The chemical structure of 1-hydroxy-6-(1-methylpropyl)-3-(2-methylproryl)-2(1H)-pyrazinone was revealed on the basis of liquid and gas mass spectroscopies. This compound should be completely safe based on toxic test results using model mice.