172 research outputs found
Visualizing spatiotemporal dynamics of apoptosis after G1 arrest by human T cell leukemia virus type 1 Tax and insights into gene expression changes using microarray-based gene expression analysis.
BACKGROUND: Human T cell leukemia virus type 1 (HTLV-1) Tax is a potent activator of viral and cellular gene expression that interacts with a number of cellular proteins. Many reports show that Tax is capable of regulating cell cycle progression and apoptosis both positively and negatively. However, it still remains to understand why the Tax oncoprotein induces cell cycle arrest and apoptosis, or whether Tax-induced apoptosis is dependent upon its ability to induce G1 arrest. The present study used time-lapse imaging to explore the spatiotemporal patterns of cell cycle dynamics in Tax-expressing HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator, Fucci2. A large-scale host cell gene profiling approach was also used to identify the genes involved in Tax-mediated cell signaling events related to cellular proliferation and apoptosis.
RESULTS: Tax-expressing apoptotic cells showed a rounded morphology and detached from the culture dish after cell cycle arrest at the G1 phase. Thus, it appears that Tax induces apoptosis through pathways identical to those involved in G1 arrest. To elucidate the mechanism(s) by which Tax induces cell cycle arrest and apoptosis, regulation of host cellular genes by Tax was analyzed using a microarray containing approximately 18,400 human mRNA transcripts. Seventeen genes related to cell cycle regulation were identified as being up or downregulated \u3e 2.0-fold in Tax-expressing cells. Several genes, including SMAD3, JUN, GADD45B, DUSP1 and IL8, were involved in cellular proliferation, responses to cellular stress and DNA damage, or inflammation and immune responses. Additionally, 23 pro- and anti-apoptotic genes were deregulated by Tax, including TNFAIP3, TNFRS9, BIRC3 and IL6. Furthermore, the kinetics of IL8, SMAD3, CDKN1A, GADD45A, GADD45B and IL6 expression were altered following the induction of Tax, and correlated closely with the morphological changes observed by time-lapse imaging.
CONCLUSIONS: Taken together, the results of this study permit a greater understanding of the biological events affected by HTLV-1 Tax, particularly the regulation of cellular proliferation and apoptosis. Importantly, this study is the first to demonstrate the dynamics of morphological changes during Tax-induced apoptosis after cell cycle arrest at the G1 phase
Visualizing spatiotemporal dynamics of apoptosis after G1 arrest by human T cell leukemia virus type 1 Tax and insights into gene expression changes using microarray-based gene expression analysis
BACKGROUND: Human T cell leukemia virus type 1 (HTLV-1) Tax is a potent activator of viral and cellular gene expression that interacts with a number of cellular proteins. Many reports show that Tax is capable of regulating cell cycle progression and apoptosis both positively and negatively. However, it still remains to understand why the Tax oncoprotein induces cell cycle arrest and apoptosis, or whether Tax-induced apoptosis is dependent upon its ability to induce G1 arrest. The present study used time-lapse imaging to explore the spatiotemporal patterns of cell cycle dynamics in Tax-expressing HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator, Fucci2. A large-scale host cell gene profiling approach was also used to identify the genes involved in Tax-mediated cell signaling events related to cellular proliferation and apoptosis.
RESULTS: Tax-expressing apoptotic cells showed a rounded morphology and detached from the culture dish after cell cycle arrest at the G1 phase. Thus, it appears that Tax induces apoptosis through pathways identical to those involved in G1 arrest. To elucidate the mechanism(s) by which Tax induces cell cycle arrest and apoptosis, regulation of host cellular genes by Tax was analyzed using a microarray containing approximately 18,400 human mRNA transcripts. Seventeen genes related to cell cycle regulation were identified as being up or downregulated \u3e 2.0-fold in Tax-expressing cells. Several genes, including SMAD3, JUN, GADD45B, DUSP1 and IL8, were involved in cellular proliferation, responses to cellular stress and DNA damage, or inflammation and immune responses. Additionally, 23 pro- and anti-apoptotic genes were deregulated by Tax, including TNFAIP3, TNFRS9, BIRC3 and IL6. Furthermore, the kinetics of IL8, SMAD3, CDKN1A, GADD45A, GADD45B and IL6 expression were altered following the induction of Tax, and correlated closely with the morphological changes observed by time-lapse imaging.
CONCLUSIONS: Taken together, the results of this study permit a greater understanding of the biological events affected by HTLV-1 Tax, particularly the regulation of cellular proliferation and apoptosis. Importantly, this study is the first to demonstrate the dynamics of morphological changes during Tax-induced apoptosis after cell cycle arrest at the G1 phase
Insect-induced daidzein, formononetin and their conjugates in soybean leaves.
In response to attack by bacterial pathogens, soybean (Gylcine max) leaves accumulate isoflavone aglucones, isoflavone glucosides, and glyceollins. In contrast to pathogens, the dynamics of related insect-inducible metabolites in soybean leaves remain poorly understood. In this study, we analyzed the biochemical responses of soybean leaves to Spodoptera litura (Lepidoptera: Noctuidae) herbivory and also S. litura gut contents, which contain oral secretion elicitors. Following S. litura herbivory, soybean leaves displayed an induced accumulation of the flavone and isoflavone aglycones 4',7-dihyroxyflavone, daidzein, and formononetin, and also the isoflavone glucoside daidzin. Interestingly, foliar application of S. litura oral secretions also elicited the accumulation of isoflavone aglycones (daidzein and formononetin), isoflavone 7-O-glucosides (daidzin, ononin), and isoflavone 7-O-(6'-O-malonyl-β-glucosides) (malonyldaidzin, malonylononin). Consistent with the up-regulation of the isoflavonoid biosynthetic pathway, folair phenylalanine levels also increased following oral secretion treatment. To establish that these metabolitic changes were the result of de novo biosynthesis, we demonstrated that labeled (13C9) phenylalanine was incorporated into the isoflavone aglucones. These results are consistent with the presence of soybean defense elicitors in S. litura oral secretions. We demonstrate that isoflavone aglycones and isoflavone conjugates are induced in soybean leaves, not only by pathogens as previously demonstrated, but also by foliar insect herbivory
Metataxonomic Analysis of the Uterine Microbiota Associated with Low Fertility in Dairy Cows Using Endometrial Tissues Prior to First Artificial Insemination
The deterioration in reproductive performance in association with low fertility leads to significant economic losses on dairy farms. The uterine microbiota has begun to attract attention as a possible cause of unexplained low fertility. We analyzed the uterine microbiota associated with fertility by 16S rRNA gene amplicon sequencing in dairy cows. First, the alpha (Chao1 and Shannon) and beta (unweighted and weighted UniFrac) diversities of 69 cows at four dairy farms that had passed the voluntary waiting period before the first artificial insemination (AI) were analyzed with respect to factors including farm, housing style, feeding management, parity, and AI frequency to conception. Significant differences were observed in the farm, housing style, and feeding management, except parity and AI frequency to conception. The other diversity metrics did not show significant differences in the tested factors. Similar results were obtained for the predicted functional profile. Next, the microbial diversity analysis of 31 cows at a single farm using weighted UniFrac distance matrices revealed a correlation with AI frequency to conception but not with parity. In correlation with AI frequency to conception, the predicted function profile appeared to be slightly modified and a single bacterial taxon, Arcobacter, was detected. The bacterial associations related to fertility were estimated. Considering these, the uterine microbiota in dairy cows can be varied depending on the farm management practices and may become one of the measures for low fertility
Effect of Immunomodulatory Artificial Blood Exchange (IABX) on Guinea Pig-to-Rat Heart Xenografts
The aim of this study was to assess the effectiveness of pregraft immunomodulatory artificial blood exchange (IABX) in a guinea pig-to-rat xeno discordant heart transplantation, using an artificial blood (FC43 emulsion: The Green Cross, Japan) in exchange for a large volume of whole blood to remove humoral immune factors en bloc from the recipient rat. In the rats treated with IABX, rhythmic beating of the grafted heart was maintained for 2 hr, whereas the untreated heart beat lasted for only 15.2 ± 5.2 min (n=6). In the graft hearts treated with IABX, no pathologic changes such as multiple coronary thromboses due to hyperacute rejection (HAR) were observed. Humoral immune factors (natural-IgM titer, ACH50 and CH50 complement activities, platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen serum concentration), which are thought to contribute to HAR, decreased significantly following the IABX treatment.
We conclude that IABX is an efficient method for prolonging the survival time of guinea pig heart xenografts by inhibiting thrombus formation in the xeno-graft heart. It was confirmed that IABX could remove recipient humoral immune factors en bloc.This study was supported by The Green Cross Co.,Ltd
Hemodynamic Performance of the Biventricular Bypass System Operated in an Independent Variable Rate Mode
The present study was undertaken to determine whether a biventricular bypass system operated in an independent variable rate (VR) mode can maintain the entire circulation. Two pusher-plate pumps which incorporated the Hall effect position sensors were used to bypass the right and left ventricles in 10 sheep under fibrillation. The flow distributions of the pump output to the carotid and renal arteries were investigated every 6 h using ultrasonic blood flow meters for 24 h in 5 animals, and the controllability of the VR mode was evaluated in 5 long-term experiments. The carotid artery flow ratio to the pump output decreased significantly from 4.7 +/- 0.8% before the bypass to 2.7 +/- 0.9% after 24 h. However, the renal artery flow ratio did not change throughout the experiments. In the long-term experiments, the animals were kept alive from 3 to 48 days (mean 15.6 days). The mean pump output had been maintained at more than 90 ml/min/kg for the first 7 days. After the surgery, the pump driving conditions were not readjusted in any experiment. The results indicate that the biventricular bypass system operated in the independent VR mode automatically maintains the entire circulation at a satisfactory level.</p
Reducing the Number of Long-term Items : Optimal Strategy for a Bag-packing Problem
When several items having different weights are packed into a bag, the sum weight of each packed bag needs to be constant. The problem at packing is how to determine the items to be included in a bag so that the sum weight should be closest to the desired weight. In this paper, the following two algorithms are shown; one is to solve as an optimization problem and the other is to solve as a constraint satisfaction problem. Comparing the two algorithms, their functional rolls are made clear. In order to reduce the number of items remaining in scale hoppers for a long time, a method is proposed in which each of the two algorithms can be turned over one after another. Simulation results seem to prove that the proposed method is preferable
Contacts between the commissural axons and the floor plate cells are mediated by nectins
AbstractDuring development of the central nervous system (CNS), commissural axons grow toward the ventral midline. After crossing the floor plate, they abruptly change their trajectory from the circumferential to the longitudinal axis. The contacts between the commissural axons and the floor plate cells are involved in this axonal guidance, but their mechanisms or structures have not fully been understood. In this study, we found that nectin-1 and -3, immunoglobulin-like cell–cell adhesion molecules, asymmetrically localized at the contact sites between the commissural axons and the floor plate cells, respectively. In vitro perturbation of the endogenous trans-interaction between nectin-1 and -3 caused abnormal fasciculation of the commissural axons and impairment of the contacts, and resulted in failure in longitudinal turns of the commissural axons at the contralateral sites of the rat hindbrain. These results indicate that the contacts between the commissural axons and the floor plate cells are mediated by the hetero-trans-interaction between nectin-1 and -3 and involved in regulation of the trajectory of the commissural axons
Use of Recombinant Endolysin to Improve Accuracy of Group B Streptococcus Tests
Group B Streptococcus (GBS) causes serious neonatal infection via vertical transmission. The prenatal GBS screening test is performed at the late stage of pregnancy to avoid risks of infection. In this test, enrichment culture is performed, followed by GBS identification. Selective medium is used for the enrichment; however, Enterococcus faecalis, which is a potential contaminant in swab samples, can interfere with the growth of GBS. Such bacterial contamination can lead to false-negative results. Endolysin, a bacteriophage-derived enzyme, degrades peptidoglycan in the bacterial cell wall; it is a promising antimicrobial agent for selectively eliminating specific bacterial genera/species. In this study, we used the recombinant endolysin EG-LYS, which is specific to E. faecalis; the endolysin potentially enriched GBS in the selective culture. First, in the false-negative model (coculture of GBS and E. faecalis, which disabled GBS detection in the subsequent GBS identification test), EG-LYS treatment at 0.1 mg/ml improved GBS detection. Next, we used 548 vaginal swabs to test the efficacy of EG-LYS treatment in improving GBS detection. EG-LYS treatment (0.1 mg/ml) increased the GBS-positive ratio to 17.9%, compared to 15.7% in the control (phosphate-buffered saline [PBS] treatment). In addition, there were an increased number of GBS colonies under EG-LYS treatment in some samples. The results were supported by the microbiota analysis of the enriched cultures. In conclusion, EG-LYS treatment of the enrichment culture potentially improves the accuracy of the prenatal GBS screening test
Visualizing bovine leukemia virus (BLV)-infected cells and measuring BLV proviral loads in the milk of BLV seropositive dams
International audienceAbstractBovine leukemia virus (BLV) infects cattle and causes serious problems for the cattle industry, worldwide. Vertical transmission of BLV occurs via in utero infection and ingestion of infected milk and colostrum. The aim of this study was to clarify whether milk is a risk factor in BLV transmission by quantifying proviral loads in milk and visualizing the infectivity of milk. We collected blood and milk from 48 dams (46 BLV seropositive dams and 2 seronegative dams) from seven farms in Japan and detected the BLV provirus in 43 blood samples (89.6%) but only 22 milk samples (45.8%) using BLV-CoCoMo-qPCR-2. Although the proviral loads in the milk tended to be lower, a positive correlation was firstly found between the proviral loads with blood and milk. Furthermore, the infectivity of milk cells with BLV was visualized ex vivo using a luminescence syncytium induction assay (LuSIA) based on CC81-GREMG cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) in response to BLV Tax and Env expressions when co-cultured with BLV-infected cells. Interestingly, in addition to one BLV-infected dam with lymphoma, syncytia with EGFP fluorescence were observed in milk cells from six BLV-infected, but healthy, dams by an improved LuSIA, which was optimized for milk cells. This is the first report demonstrating the infectious capacity of cells in milk from BLV-infected dams by visualization of BLV infection ex vivo. Thus, our results suggest that milk is a potential risk factor for BLV vertical spread through cell to cell transmission
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