Fas antigen (Apo-1/CD95) is an apoptosis-signaling cell surface receptor belonging to the tumor necrosis factor receptor superfamily. Adult T cell leukemia (ATL) cells express Fas antigen and show apoptosis after treatment with an anti-Fas monoclonal antibody. We established the ATL cell line KOB, which showed resistance to Fas-mediated apoptosis, and found that KOB expressed two forms of Fas mRNA, the normal form and a truncated form. The truncated transcript lacked 20 base pairs at exon 9, resulting in a frame shift and the generation of a premature stop codon at amino acid 239. The same mutation was detected in primary ascitic cells and peripheral blood cells. The mutation was not detected in lymph node cells, however, although all of the primary ATL cells were of the same clonal origin. A retroviral-mediated gene transfer of the truncated Fas to Jurkat cells rendered the cells resistant to Fas-mediated apoptosis, suggesting a dominant negative interference mechanism. These results indicate that an ATL subclone acquires a Fas mutation in the lymph nodes, enabling the subclone to escape from apoptosis mediated by the Fas/Fas ligand system and proliferate in the body. Mutation of the Fas gene may be one of the mechanisms underlying the progression of ATL.

Immunotherapeutic approaches, particularly programmed death 1/programmed death ligand 1 (PD-1/PD-L1) blockade, have improved the treatment of non-small-cell lung cancer (NSCLC), supporting the premise that evasion of immune destruction is of importance for NSCLC progression. However, the signals responsible for upregulation of PD-L1 in NSCLC cells and whether they are integrated with the regulation of other immune-related genes are not known. Mucin 1 (MUC1) is aberrantly overexpressed in NSCLC, activates the nuclear factor-κB (NF-κB) p65→︀ZEB1 pathway and confers a poor prognosis. The present studies demonstrate that MUC1-C activates PD-L1 expression in NSCLC cells. We show that MUC1-C increases NF-κB p65 occupancy on the CD274/PD-L1 promoter and thereby drives CD274 transcription. Moreover, we demonstrate that MUC1-C-induced activation of NF-κB→︀ZEB1 signaling represses the TLR9 (toll-like receptor 9), IFNG, MCP-1 (monocyte chemoattractant protein-1) and GM-CSF genes, and that this signature is associated with decreases in overall survival. In concert with these results, targeting MUC1-C in NSCLC tumors suppresses PD-L1 and induces these effectors of innate and adaptive immunity. These findings support a previously unrecognized central role for MUC1-C in integrating PD-L1 activation with suppression of immune effectors and poor clinical outcome.

Tissue responses to experimentally induced actinomycotic lesions were investigated in mice by both light and transmission electron microscopy. Micro-organisms of Actinomyces israelii were entrapped in alginate gel and injected into the subcutaneous tissue over the periosteum of the mouse cranium. One day after the injection (initial stage), a non-stained amorphous structure was located in the core of the lesion, corresponding to the injected gel with bacteria. Numerous neutrophils surrounded the core region and phagocytized the injected complex actively. At days 3-7 (intermediate stage), the lesion became well developed. The core structure became eosinophilic and separated to form island-like structures. No lesion was recognized in the control group (gel without bacteria) until day 14. After 30 days (late stage), the lesions displayed more static features, similar to the "sulphur granules" characteristic of actinomycotic lesions. At the late stage, foamy cells increased in number and took the place of neutrophils in the alginate islands. By transmission electron microscopy these foamy cells were seen to be filled with lysosomal vesicles containing electron-dense foreign material. Thus, these cells appeared to be macrophages that had phagocytized degenerated neutrophils containing bacteria. Along with the active phagocytosis by foamy cells that progressed in the late stage, a collagenous capsule became conspicuous and separated the lesion from the intact tissue. The bacteria remained in the gel islands until at least day 60, although they considerably decreased in number with time. Serum IgG antibody titres began to rise within 24 h of the injection, reached a peak concentration at day 14 and remained a significantly high (p < 0.01, vs 0 time) until day 120. These results suggest that this animal model is useful for inducing experimental chronic infectious lesions.

The effects of MPC-1304, a newly developed 1,4-dihydropyridine derivative, on blood pressure and hypertensive complications in stroke-prone spontaneously hypertensive rats fed a high-salt diet (0.8% NaCl), were investigated. The antihypertensive effectiveness of nicardipine was used for the purpose of comparison. MPC-1304 and nicardipine were added to the diet, in doses of 0.01% (0.01% MPC-1304 diet), 0.03% (0.03% MPC-1304 diet) and 0.1% (0.1% nicardipine diet), respectively, throughout the experimental period (8 to 30 weeks of age). This chronic ingestion of MPC-1304 and nicardipine inhibited the development of hypertension and reduced the concentration of blood urea nitrogen, creatinine, triglyceride and total cholesterol in serum. Treatment with MPC-1304 inhibited the incidence of cerebral stroke, cardiac fibrosis, proliferative and fibrinoid arteriolitis and malignant nephrosclerosis. There was no significant difference in the antihypertensive effectiveness between 0.01% MPC-1304 and 0.1% nicardipine diets. Thus, MPC-1304 had antihypertensive effects in stroke-prone spontaneously hypertensive rats.

Epithelial ovarian carcinoma is thought to derive from ovarian surface epithelium (OSE). The black box of the early molecular changes in ovarian carcinogenesis is being interpreted by the development of experimental systems employing immortalised human OSE cells. However, the existing cell lines of the OSE cells have limited utility due to chromosomal instability. Our goal was to establish new immortalised human OSE cells that retain the original characteristics of the primary cells without chromosomal alterations. Using primary human OSE cells obtained from a postmenopausal patient with endometrial cancer, five cell lines ('HOSE1' lines) were newly established by infection with retroviral expression vectors containing type 16 human papillomavirus (HPV-16) E6, E7, a variant E6 (E6delta151), and Bmi1 polycomb gene, in combination with telomerase reverse transcriptase (hTERT). Consequently, five HOSE1s cell lines, HOSE1s-E6/hTERT, -E7/hTERT, -E6/E7/hTERT, -E6delta151/E7/hTERT, and -E6delta151/Bmi1/hTERT, grew beyond the population doubling number of 200. These cell lines, except for HOSE1-E6/hTERT, essentially showed the original features of the primary human OSE cells. Of them, HOSE1-E7/hTERT preserved diploidy in a kariotype analysis, and did not show transformed phenotypes in anchorage-independent growth and tumour formation. Thus, HOSE1-E7/hTERT may provide a novel model system with which to investigate the mechanisms of early molecular changes.

We reported a case of malignant rheumatoid arthritis (MRA) with cerebral infarction associated with a possible cause of lupus anticoagulant. The patient was a 68-year-old woman who had received treatment for rheumatoid arthritis (RA) from 15 to 16 years ago. She consulted to our hospital with a major complaint of right hemiplegia. Brain CT revealed a low density area in the left hemisphere. She was diagnosed as cerebral infarction and hospitalized. Since she was noted to have hypocomplementemia, interstitial pneumonia and pericarditis, she was diagnosed as MRA. Coagulation test disclosed positive lupus anticoagulant (LA). Generally, CNS disorders in MRA are uncommon. Cerebral infarction was complicated in the present case, suggesting the involvement of antiphospholipid antibodies as its pathogenesis.

MAPK (mitogen-activated protein kinase) cascades are common eukaryotic signaling modules that consist of a MAPK, a MAPK kinase (MAPKK) and a MAPKK kinase (MAPKKK). Because phosphorylation is essential for the activation of both MAPKKs and MAPKs, protein phosphatases are likely to be important regulators of signaling through MAPK cascades. To identify protein phosphatases that negatively regulate the stress-responsive p38 and JNK MAPK cascades, we screened human cDNA libraries for genes that down-regulated the yeast HOG1 MAPK pathway, which shares similarities with the p38 and JNK pathways, using a hyperactivating yeast mutant. In this screen, the human protein phosphatase type 2Calpha (PP2Calpha) was found to negatively regulate the HOG1 pathway in yeast. Moreover, when expressed in mammalian cells, PP2Calpha inhibited the activation of the p38 and JNK cascades induced by environmental stresses. Both in vivo and in vitro observations indicated that PP2Calpha dephosphorylated and inactivated MAPKKs (MKK6 and SEK1) and a MAPK (p38) in the stress-responsive MAPK cascades. Furthermore, a direct interaction of PP2Calpha and p38 was demonstrated by a co-immunoprecipitation assay. This interaction was observed only when cells were stimulated with stresses or when a catalytically inactive PP2Calpha mutant was used, suggesting that only the phosphorylated form of p38 interacts with PP2Calpha.

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.

Alterations in the levels of growth-associated protein 43 (GAP-43)-like immunoreactivity (-LI) were examined in the lingual periodontal ligament of the rat incisor following two types of injury (resection and crush) to the inferior alveolar nerve (IAN). In normal animals, GAP-43-like immunoreactive (IR) structures were observed as tree-like ramifications in the alveolar half of the lingual periodontal ligament of incisors. Under immunoelectron microscopy, GAP-43-LI appeared in the Schwann sheaths associated with periodontal Ruffini endings; neither cell bodies of the terminal Schwann cells nor axonal profiles showed GAP-43-LI. During regeneration of the periodontal Ruffini endings following resection of the IAN, GAP-43-LI appeared in the cytoplasm of the terminal Schwann cell bodies and axoplasm of the terminals. The distribution of GAP-43-LI in the Ruffini endings returned to almost normal levels on days 28 and 56 following the injury. The changes in the distribution of GAP-43-LI following the crush injury were similar to those following resection; however, expression of GAP-43-LI was slightly higher for the entire experimental period compared with the resection. The transient expression of GAP-43 in the terminal Schwann cells and axonal profiles of the periodontal Ruffini endings following nerve injury suggests that GAP-43 is closely associated with axon-Schwann cells interactions during regeneration.

This study examined whether the forkhead transcription factors of O group 1 (FoxO1) might be involved in telomere biology during calorie restriction (CR). We used FoxO1-knockout heterozygous mice (FoxO1(+/-)) and wild-type mice (WT) as a control. Both WT and FoxO1(+/-) were subjected to ad libitum (AL) feeding or 30% CR compared to AL for 20 weeks from 15 weeks of age. The heart-to-body weight ratio, blood glucose, and serum lipid profiles were not different among all groups of mice at the end of the study. Telomere size was significantly lower in the FoxO1(+/-)-AL than the WT-AL, and telomere attrition was not observed in either WT-CR or FoxO1(+/-)-CR. Telomerase activity was elevated in the heart and liver of WT-CR, but not in those of FoxO1(+/-)-CR. The phosphorylation of Akt was inhibited and Sirt 1 was activated in heart tissues of WT-CR and FoxO1(+/-)-CR. However, the ratio of conjugated to cytosolic light chain 3 increased and the level of p62 decreased in WT-CR, but not in FoxO1(+/-)-CR. A marker of oxidative DNA damage, 8-OhdG, was significantly lower in WT-CR only. The level of MnSOD and eNOS increased, and the level of cleaved caspase-3 decreased in WT-CR, but not FoxO1(+/-)-CR. Echocardiography showed that the left ventricular end-diastolic and systolic dimensions were significantly lower in WT-CR or FoxO1(+/-)-CR than WT-AL or FoxO1(+/-)-AL, respectively. The present studies suggest that FoxO1 plays beneficial roles by inducing genes involved in telomerase activity, as well as anti-oxidant, autophagic, and anti-apoptotic genes under conditions of CR, and suggest that FoxO1 signaling may be an important mediator of metabolic equilibrium during CR.

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is a component of the polycomb repressive complex 1 (PRC1) complex that is overexpressed in breast and other cancers, and promotes self-renewal of cancer stem-like cells. The oncogenic mucin 1 (MUC1) C-terminal (MUC1-C) subunit is similarly overexpressed in human carcinoma cells and has been linked to their self-renewal. There is no known relationship between MUC1-C and BMI1 in cancer. The present studies demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism in breast and other cancer cells. In addition, we show that MUC1-C blocks miR-200c-mediated downregulation of BMI1 expression. The functional significance of this MUC1-C→︀BMI1 pathway is supported by the demonstration that targeting MUC1-C suppresses BMI1-induced ubiquitylation of H2A and thereby derepresses homeobox HOXC5 and HOXC13 gene expression. Notably, our results further show that MUC1-C binds directly to BMI1 and promotes occupancy of BMI1 on the CDKN2A promoter. In concert with BMI1-induced repression of the p16INK4a tumor suppressor, we found that targeting MUC1-C is associated with induction of p16INK4a expression. In support of these results, analysis of three gene expresssion data sets demonstrated highly significant correlations between MUC1-C and BMI1 in breast cancers. These findings uncover a previously unrecognized role for MUC1-C in driving BMI1 expression and in directly interacting with this stem cell factor, linking MUC1-C with function of the PRC1 in epigenetic gene silencing.

With the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), a high-speed and convenient detection technology should be at the forefront of medical care worldwide. This study evaluated the usefulness of GeneSoC, a compact, high-speed reciprocal flow quantitative reverse transcription polymerase chain reaction system, for the detection of SARS-CoV-2. The results support the use of this system for the rapid identification of SARS-CoV-2. This approach can contribute to the strategic selection of initial management strategies for patients with COVID-19.

To determine whether dopamine release derived from exogenous l-DOPA is under inhibitory control of presynaptic dopamine D2 receptors in the dopaminergic denervated striatum, extracellular dopamine levels were measured in the striatum of 6-hydroxydopamine-lesioned rats using in vivo brain microdialysis. Quinpirole, a D2 agonist, dose-dependently (0.01-3 mg/kg s.c.) inhibited endogenous dopamine release both in the intact and dopaminergic denervated striatum. The dose-response curve obtained from the denervated striatum showed a shift to the right. Administration of l-DOPA (30 mg/kg i.p.) with carbidopa (25 mg/kg i.p.) increased dopamine release to 130% of basal levels in the intact striatum and 770% of basal levels in the denervated striatum. In the intact striatum, dopamine release was continuously inhibited by quinpirole pretreatment (1 mg/kg s.c.) even after l-DOPA administration. In the denervated striatum, l-DOPA-derived dopamine release was not affected by quinpirole pretreatment. These results suggest that, in the striatum with dopaminergic denervation, regulation by presynaptic D2 receptors is still operative on endogenous dopamine release but it does not work on dopamine release derived from exogenously administered l-DOPA.