12-O-Tetradecanoylphorbol-13-acetate Induces Epstein–Barr Virus Reactivation via NF-κB and AP-1 as Regulated by Protein Kinase C and Mitogen-Activated Protein Kinase

AbstractSignaling pathway components mediating Epstein–Barr virus (EBV) reactivation by 12-O-tetradecanoylphorbol-13-acetate (TPA) were characterized in terms of induction and modification of specific transacting factors. The consequences of protein kinase C (PKC) activation by TPA in inhibiting inducible nitric oxide synthase (iNOS) mRNA expression were analyzed in the EBV-infected gastric epithelial cell line GT38. Spontaneous expression of the EBV BZLF1 gene product ZEBRA became undetectable upon long-term culturing of GT38 cells, while iNOS mRNA expression increased. In such cells the PKC inhibitors 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine (H7) and staurosporine inhibited TPA-induced expression of BZLF1 and BRLF1 and reversed TPA-mediated inhibition of iNOS gene expression. The mitogen-activated protein kinase inhibitor PD98059 inhibited TPA-induced BZLF1 expression. Electrophoretic mobility shift assays demonstrated that transcription factors NF-κB and AP-1 were also activated by TPA in a time-dependent manner. The TPA-induced NF-κB activation was inhibited by prior treatment of the cells with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). TPA-induced BZLF1 expression was also inhibited by the treatment with PDTC. Northern blot analyses characterized changes in levels of the c-jun and junB expressions of the AP-1 family. These results show that TPA induces EBV reactivation via NF-κB and AP-1 and that PKC is an important mediator in regulating gene expression leading to EBV reactivation after TPA treatment of GT38 cells

Response speed control of helicity inversion based on a “regulatory enzyme”-like strategy

In biological systems, there are many signal transduction cascades in which a chemical signal is transferred as a series of chemical events. Such successive reaction systems are advantageous because the efficiency of the functions can be finely controlled by regulatory enzymes at an earlier stage. However, most of artificial responsive molecules developed so far rely on single-step conversion, whose response speeds have been difficult to be controlled by external stimuli. In this context, developing artificial conversion systems that have a regulation step similar to the regulatory enzymes has been anticipated. Here we report a novel artificial two-step structural conversion system in which the response speed can be controlled based on a regulatory enzyme-like strategy. In this system, addition of fluoride ion caused desilylation of the siloxycarboxylate ion attached to a helical complex, resulting in the subsequent helicity inversion. The response speeds of the helicity inversion depended on the reactivity of the siloxycarboxylate ions; when a less-reactive siloxycarboxylate ion was used, the helicity inversion rate was governed by the desilylation rate. This is the first artificial responsive molecule in which the overall response speed can be controlled at the regulation step separated from the function step

Regulation of the Expression of Epidermal Growth Factor Receptor mRNA with Thyroid Hormone L-3,5,3'-Triiodothyronine in Rat Hepatoma Cells

The expression of epidermal growth factor (EGF) receptor mRNA was examined using thyroid hormone L-3,5,3'-triiodothyronine (T3) by Northern blot analysis in rat hepatoma cells. The expression of full-length, but not truncated receptor mRNA was enhanced and destabilized with T3. These results suggest that T3 induces not only the expression of full-length EGF receptor mRNA but also accelerates their rate of turnover. The 3' terminal region of full-length EGF receptor mRNA may have a role in the destabilization of EGF receptor mRNA by T3, because truncated EGF receptor mRNA lacks only the 3' terminal region of full-length EGF receptor mRNA

Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells

Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine-containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti-Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells

Increased Activity of 2',5'-Oligoadenylate Synthetase in Peripheral Blood Mononuclear Cells of Patients with Major Depressive Disorder

The aim of this study was to measure the activity of 2',5'-oligoadenylate synthetase (2',5'AS) which is mainly induced by interferon (IFN)-α and -β in patients with major depression and evaluate its relationship to the disease. 2',5'AS activity of 23 patients (male = 11, female = 12) with major depression and of 29 normal control subjects (male = 15, female = 14) was measured in peripheral blood mononuclear cells (PBMCs) by radioassay. The mean 2',5'AS activity in the PBMCs of the patients and that of the control subjects were 1.10 ± 0.69% and 0.72 ± 0.51%, respectively. The activity in the patients was statistically higher than that of the control subjects (P = 0.03). These results imply some abnormality in the IFN-2',5'AS system of patients with major depression

Tumorigenesis of Epstein–Barr Virus-Positive Epithelial Cell Lines Derived from Gastric Tissues in the SCID Mouse

AbstractTo study the tumorigenesis of Epstein–Barr virus (EBV)-positive epithelial cell lines GT38 and GT39 derived from human gastric tissues, we inoculated these cells under the skin of severe combined immunodeficient (SCID) mice. The development of tumors was observed in each of the mice about 2 months after the inoculation. The tumors were diagnosed with undifferentiated carcinoma by hematoxylin/eosin staining. EBV-encoded small RNA1 was detected in the paraffin-embedded tumor sections. The tumor cells had human chromosome. The circular, but not linear, EBV DNA was detected in the tumors. The molecular sizes of EBV DNA termini were the same as that of the inoculated GT38 or GT39 cells. The expressions of EBV nuclear antigen 2 and latent membrane protein 1 reduced in the tumors. Transcripts of BamHI C and W promoters in latency III were detected in the tumors and the cultured cells in vitro. The tumor cells were passaged from one SCID mouse to other SCID mice and to cultures in vitro. This is the first evidence that the EBV-positive epithelial cell lines produced tumors in the SCID mouse

Absolute Configuration Determination from Low ee Compounds by the Crystalline Sponge Method. Unusual Conglomerate Formation in a Pre-Determined Crystalline Lattice

When chiral compounds with low enantiomeric excess (ee, R:S=m:n) were absorbed into the void of the crystalline sponge (CS), enantiomerically pure [(R)m(S)n] chiral composites were formed, changing the centrosymmetric space group into non-centrosymmetric one. The absolute configuration of the analyte compounds was elucidated with a reasonable Flack (Parsons) parameter value. This phenomenon is characteristic to the “post-crystallization” in the pre-determined CS crystalline lattice, seldom found in common crystallization where the crystalline lattice is defined by an analyte itself. The results highlight the potential of the CS method for absolute configuration determination of low ee samples, an often encountered situation in asymmetric synthesis studies

マツモト ミツオ センセイ ゴリャクレキ

松本充郎准教授追悼特集In Honor of the Memory of Professor Mitsuo MATSUMOT

A Novel Curriculum for Medical Student Training in LGBTQ Healthcare: A Regional Pathway Experience.

Background: Lesbian, gay, bisexual, transgender, and queer (LGBTQ) individuals face considerable health disparities, often due to a lack of LGBTQ-competent care. Such disparities and lack of access to informed care are even more staggering in rural settings. As the state medical school for the Washington, Wyoming, Alaska, Montana, and Idaho (WWAMI) region, the University of Washington School of Medicine (UWSOM) is in a unique position to train future physicians to provide healthcare that meets the needs of LGBTQ patients both regionally and nationally. Objective: To describe our methodology of developing a student-driven longitudinal, region-wide curriculum to train medical students to provide high-quality care to LGBTQ patients. Methods: A 4-year LGBTQ Health Pathway was developed and implemented as a student-led initiative at the UWSOM. First- and second-year medical students at sites across the WWAMI region are eligible to apply. Accepted Pathway students complete a diverse set of pre-clinical and clinical components: online modules, didactic courses, longitudinal community service/advocacy work, a scholarly project, and a novel clinical clerkship in LGBTQ health developed specifically for this Pathway experience. Students who complete all requirements receive a certification of Pathway completion. This is incorporated into the Medical Student Performance Evaluation as part of residency applications. Results: The LGBTQ Health Pathway is currently in its fourth year. A total of 43 total students have enrolled, of whom 37.3% are based in the WWAMI region outside of Seattle. Pathway students have completed a variety of scholarly projects on LGBTQ topics, and over 1000 hours of community service/advocacy. The first cohort of 8 students graduated with a certificate of Pathway completion in spring 2020. Conclusions: The LGBTQ Health Pathway at UWSOM is a novel education program for motivated medical students across the 5-state WWAMI region. The diverse milestones, longitudinal nature of the program, focus on rural communities, and opportunities for student leadership are all strengths and unique aspects of this program. The Pathway curriculum and methodology described here serve as a model for student involvement and leadership in medical education. This program enables medical students to enhance their training in the care of LGBTQ patients and provides a unique educational opportunity for future physicians who strive to better serve LGBTQ populations