Genes to Cells最新文献

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

The hydrogen peroxide (H₂O₂)-producing NADPH oxidase Nox4, forming a heterodimer with p22^phox, is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H₂O₂ production is controlled by its protein abundance. Hypoxia-induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level. Here we show that hypoxia upregulates the Nox4 protein level and Nox4-catalyzed H₂O₂ production without increasing the Nox4 mRNA in rat H9c2 cardiomyocytes. In these cells, the Nox4 protein is stabilized under hypoxic conditions in a manner dependent on the presence of p22^phox. Cell treatment with the proteasome inhibitor MG132 results in a marked decrease of the Nox4 protein under both normoxic and hypoxic conditions, indicating that the proteasome pathway does not play a major role in Nox4 degradation. The decrease is partially restored by the autophagy inhibitor 3-methyladenine. Furthermore, the Nox4 protein level is upregulated by the lysosome inhibitors bafilomycin A1 and chloroquine. Thus, in cardiomyocytes, Nox4 appears to be degraded via an autophagy-related pathway, and its suppression by hypoxia likely stabilizes Nox4, leading to upregulation of Nox4-catalyzed H₂O₂ production.

The mitotic cohesin complex necessary for sister chromatid cohesion and chromatin loop formation shows local and global association to chromosomes in response to DNA double-strand breaks (DSBs). Here, by genome-wide binding analysis of the meiotic cohesin with Rec8, we found that the Rec8-localization profile along chromosomes is altered from middle to late meiotic prophase I with cleavage-independent dissociation. Each Rec8-binding site on the chromosome axis follows a unique alternation pattern with dissociation and probably association. Centromeres showed altered Rec8 binding in late prophase I relative to mid-prophase I, implying chromosome remodeling of the regions. Rec8 dissociation ratio per chromosome is correlated well with meiotic DSB density. Indeed, the spo11 mutant deficient in meiotic DSB formation did not change the distribution of Rec8 along chromosomes in late meiotic prophase I. These suggest the presence of a meiosis-specific regulatory pathway for the global binding of Rec8-cohesin in response to DSBs.

The c-Jun N-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein of mitogen-activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non-transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non-transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP-overexpressed cells. Moreover, we established JLP-inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild-type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild-type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression.

Glyoxalase I (GLO I), a major enzyme involved in the detoxification of the anaerobic glycolytic byproduct methylglyoxal, is highly expressed in various tumors, and is regarded as a promising target for cancer therapy. We recently reported that piceatannol potently inhibits human GLO I and induces the death of GLO I-dependent cancer cells. Pyruvate kinase M2 (PKM2) is also a potential therapeutic target for cancer treatment, so we evaluated the combined anticancer efficacy of piceatannol plus low-dose shikonin, a potent and specific plant-derived PKM2 inhibitor, in two GLO I-dependent cancer cell lines, HL-60 human myeloid leukemia cells and NCI-H522 human non-small-cell lung cancer cells. Combined treatment with piceatannol and low-dose shikonin for 48 h synergistically reduced cell viability, enhanced apoptosis rate, and increased extracellular methylglyoxal accumulation compared to single-agent treatment, but did not alter PKM1, PKM2, or GLO I protein expression. Taken together, these results indicate that concomitant use of low-dose shikonin potentiates piceatannol-induced apoptosis of GLO I-dependent cancer cells by augmenting methylglyoxal accumulation.

Avians have thrombocytes in their blood circulation rather than mammalian platelets. However, many details of thrombocyte characteristics have not been determined. Here, chicken thrombocytes were isolated, and extracellular vesicle (EV) production was investigated. The thrombocyte-specific markers cd41 and cd61 were expressed in the yolk sac at 24 h. According to the embryonic developmental stage, the cd41-expressing tissues changed from the yolk sac to the bone marrow and spleen. Accordingly, the bone marrow and spleen were the main tissues producing thrombocytes in adult chickens. Avian thrombocytes were separated from adult spleen cells through a combination of discontinuous density gradient centrifugation, phagocytic cell removal, and fluorescence-activated cell sorting. Isolated thrombocytes produced CD41⁺ EVs (CD41⁺ EVs), and the CD41⁺ EVs also expressed CD9. Microarray analysis revealed that CD41⁺ EVs contain many microRNAs. Macrophage lines (RAW264.7) phagocytosed CD41⁺ EVs, and their phagocytosis and migration activity were suppressed. Microarray analysis also revealed that EVs altered gene expression in macrophages. These data indicated that the CD41⁺ EV was a carrier of microRNAs produced from thrombocytes and affected the cell characteristics of the received cells. Therefore, the CD41⁺ EVs of avians worked as a communication tool.

One hallmark of some autoimmune diseases is the variability of symptoms among individuals. Organs affected by the disease differ between patients, posing a challenge in diagnosing the affected organs. Although numerous studies have investigated the correlation between T cell antigen receptor (TCR) repertoires and the development of infectious and immune diseases, the correlation between TCR repertoires and variations in disease symptoms among individuals remains unclear. This study aimed to investigate the correlation of TCRα and β repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and β of CD4⁺CD44^highCD62L^low T cells in the blood and stomachs of mice deficient in autoimmune regulator (Aire) (AIRE KO), a mouse model of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Data analysis revealed that the degree of similarity in TCR sequences between the blood and stomach varied among individual AIRE KO mice and reflected the extent of T cell infiltration in the stomach. We identified a set of TCR sequences whose frequencies in blood might correlate with extent of the stomach manifestations. Our results propose a potential of using TCR repertoires not only for diagnosing disease development but also for diagnosing affected organs in autoimmune diseases.

A simple method for producing pseudopregnant mice supports pup production. In this study, pregnant ICR were obtained mice without mating with vasectomized mice via administration of mouse Kisspeptin-10 (mKp-10) and transferring blastocysts to the uterus. Blastocyst transfer after mKp-10 administration to mice with gapping and reddish pink vagina resulted in 65.2% (15/23) pregnancies, and 39.1% (34/87) of the transferred blastocysts showed full-term growth. Vaginal smears were observed for accurate estrus cycle determination, and subsequent administration of mKp10 to mice during the estrus stage and blastocyst transfer resulted in 95.2% (20/21) pregnancies and 50.7% (104/205) birth rates. Regarding 2-cell transfer after administration of mKp-10, 100% (8/8) of the mice became pregnant, and 45.0% (36/80) of the embryos were born. Administration of mKp-10 to mice during the estrus stage is a convenient way to generate pseudopregnant mice.

The transcriptome data of skin cells from domestic cats with brown, orange, and white coats were analyzed using a public database to investigate the possible relationship between coat color-related gene expression and squamous cell carcinoma risk, as well as the mechanism of deafness in white cats. We found that the ratio of the expression level of genes suppressing squamous cell carcinoma to that of genes promoting squamous cell carcinoma might be considerably lower than the theoretical estimation in skin cells with orange and white coats in white-spotted cat. We also found the possibility of the frequent production of KIT lacking the first exon (d1KIT) in skin cells with white coats, and d1KIT production exhibited a substantial negative correlation with the expression of SOX10, which is essential for melanocyte formation and adjustment of hearing function. Additionally, the production of d1KIT was expected to be due to the insulating activity of the feline endogenous retrovirus 1 (FERV1) LTR in the first intron of KIT by its CTCF binding sequence repeat. These results contribute to basic veterinary research to understand the relationship between cat skin coat and disease risk, as well as the underlying mechanism.

The internalization of engineered high-density lipoprotein nanoparticles (engineered lipoproteins [eLPs]) with different lipid and protein compositions, zeta potentials, and/or sizes were analyzed in representative plant and mammalian cells. The impact of the addition of a cell-penetrating peptide to eLPs on the internalization was very small in Bright Yellow (BY)-2 protoplasts compared with HeLa cells. When eLPs were prepared with one of the abundant lipids in BY-2 cells, digalactosyldiacylglycerol (DGDG) (eLP4), its internalization was dramatically increased only in HeLa cells. Such an increase in HeLa cells was also obtained for liposomes containing DGDG in a DGDG content-dependent manner. Increasing the size and zeta potential of eLPs improved their internalization in both HeLa cells and in BY-2 protoplasts but to quite varying degrees. Although eLPs tended to stay at the plasma membrane (PM) in BY-2 protoplasts with much less internalization, the PM-bound eLPs somehow promoted the internalization of coexisting nanobeads in cell culture media. These results provide fundamental insight into the future design of lipid nanoparticles for drug delivery in mammalian and plant cells.