Covalently bound flavin in the NqrB and NqrC subunits of Na+-translocating NADH-quinone reductase from Vibrio alginolyticus

AbstractNa+-translocating NADH-quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS–PAGE of the purified complex, NqrB and NqrC subunits were found to give yellow–green fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed-phase high performance liquid chromatography. N-Terminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin-linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress

Internet use and problematic Internet use among adolescents in Japan: A nationwide representative survey

AbstractIntroductionJapan is assumed to have serious health and social problems due to Internet overuse, but little is known about the actual conditions. This study was conducted to investigate the prevalence of problematic Internet use (PIU) and associated Internet use in adolescents in Japan.MethodsA nationwide survey of adolescent Internet use was conducted in 2012 and 2013. The participants were 100,050 students from randomly selected junior and senior high schools nationwide who gave valid responses to a self-reported questionnaire. The questionnaire included questions on Internet use and the Japanese version of the Young's Diagnostic Questionnaire (YDQ) as well as other questions related to lifestyle habits. Internet users were classified by gender according to three categories on the basis of their YDQ scores: adaptive use, maladaptive use, and PIU.ResultsThe estimated prevalence of PIU was 6.2% in males, 9.8% in females, and 7.9% in total; it closely correlated with female gender, school grades, and number of Internet hours. The following common and gender-specific applications that conferred a risk of PIU were identified: downloading (both genders), online gaming (males), social networking services, blogs, and bulletin boards (females).ConclusionsA cross-sectional survey using YDQ of a large number of high school students yielded a PIU prevalence of 7.9% in Japan. This study showed that problems associated with Internet overuse have already become serious; therefore, planning and implementation of prevention and control measures is urgently required

Proinsulin-Reactive CD4 T Cells in the Islets of Type 1 Diabetes Organ Donors

Proinsulin is an abundant protein that is selectively expressed by pancreatic beta cells and has been a focus for development of antigen-specific immunotherapies for type 1 diabetes (T1D). In this study, we sought to comprehensively evaluate reactivity to preproinsulin by CD4 T cells originally isolated from pancreatic islets of organ donors having T1D. We analyzed 187 T cell receptor (TCR) clonotypes expressed by CD4 T cells obtained from six T1D donors and determined their response to 99 truncated preproinsulin peptide pools, in the presence of autologous B cells. We identified 14 TCR clonotypes from four out of the six donors that responded to preproinsulin peptides. Epitopes were found across all of proinsulin (insulin B-chain, C-peptide, and A-chain) including four hot spot regions containing peptides commonly targeted by TCR clonotypes derived from multiple T1D donors. Of importance, these hot spots overlap with peptide regions to which CD4 T cell responses have previously been detected in the peripheral blood of T1D patients. The 14 TCR clonotypes recognized proinsulin peptides presented by various HLA class II molecules, but there was a trend for dominant restriction with HLA-DQ, especially T1D risk alleles DQ8, DQ2, and DQ8-trans. The characteristics of the tri-molecular complex including proinsulin peptide, HLA-DQ molecule, and TCR derived from CD4 T cells in islets, provides an essential basis for developing antigen-specific biomarkers as well as immunotherapies

Tuberous Sclerosis 2 Gene Is Expressed at High Levels in Specific Types of Neurons in the Mouse Brain

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by mental retardation, epilepsy and hamartomatous growth in many tissues. The gene (TSC2) encoding a tumor suppressor protein whose mutations cause TSC, has been demonstrated to be expressed at high levels in the adult and developing brain, raising the question of whether or not the TSC2 gene product has unique roles in differentiation related to cytoskeletal interactions within the central nervous system, in addition to a tumor suppressor function. To determine the expression of TSC2 in functionally distinct neuron types of the mouse brain, we carried out in situ hybridization with digoxigenin-labeled riboprobes for the detection of TSC2 mRNA. High levels of the TSC2 gene were in neurons of the pyramidal and dentate granular layer in the hippocampus, cerebellar Purkinje cells, neurons of the piriform cortex, motor neurons in the medulla and interneurons in the striatum, while intermediate levels were in cortical neurons, striatal neurons, septal neurons, thalamic neurons and neurons in the substantia nigra compacta. Thus, the high expression of the TSC2 gene has restricted distribution in specific neuronal types which are characterized by well-developed dendrites and rich in use-dependent long-term changes in synaptic efficacy. These results suggest that the function of the TSC2 gene product may be involved on a cellular basis in neuronal plasticity and relevant to mental retardation observed in TSC patients

Electrified Fuzzy Spheres and Funnels in Curved Backgrounds

We use the non-Abelian DBI action to study the dynamics of $N$ coincident $Dp$-branes in an arbitrary curved background, with the presence of a homogenous world-volume electric field. The solutions are natural extensions of those without electric fields, and imply that the spheres will collapse toward zero size. We then go on to consider the $D1-D3$ intersection in a curved background and find various dualities and automorphisms of the general equations of motion. It is possible to map the dynamical equation of motion to the static one via Wick rotation, however the additional spatial dependence of the metric prevents this mapping from being invertible. Instead we find that a double Wick rotation leaves the static equation invariant. This is very different from the behaviour in Minkowski space. We go on to construct the most general static fuzzy funnel solutions for an arbitrary metric either by solving the static equations of motion, or by finding configurations which minimise the energy. As a consistency check we construct the Abelian $D3$-brane world-volume theory in the same generic background and find solutions consistent with energy minimisation. In the $NS$5-brane background we find time dependent solutions to the equations of motion, representing a time dependent fuzzy funnel. These solutions match those obtained from the $D$-string picture to leading order suggesting that the action in the large $N$ limit does not need corrections. We conclude by generalising our solutions to higher dimensional fuzzy funnels.Comment: 38 pages, Latex; references adde

Gingival bleeding and pocket depth among smokers and the related changes after short-term smoking cessation

Background: Smoking is associated with the deteriorating health of the gingiva and periodontium. The long-term beneficial effects of smoking cessation on oral health are well known. However, the effects of short-term smoking cessation on gingival bleeding and periodontal pocket depth are unknown. The purpose of the present study was to determine the effects of short-term smoking cessation on gingival bleeding and periodontal pocket depth. Methods: Dentate smokers with a mean age of 56.9 ± 14.4 years at an outpatient smoking cessation clinic participated in this study. A professional dentist checked the periodontal pocket depth and gingival bleeding. Patients visited the smoking cessation clinic on their first visit and 2, 4, 8, and 12 weeks (three months). The gingival assessment was re-performed in those who succeeded in smoking cessation 3 months after the baseline. Results: The baseline data of 83 patients showed that an increase in pocket depth was associated with increasing age and the amount of smoking. A significant increase in gingival bleeding (p = .031) and increase in pocket depth (p = .046) were observed 3 months after the baseline in patients who successfully quit smoking (n = 14). Conclusion: Short-term smoking cessation increased periodontal pocket depth and gingival bleeding. These findings may reflect healing processes that occur in the healthy gingiva. Implications: Study findings will be useful to advise patients during smoking cessation programs. Dentists can inform patients that an initial increase in gingival bleeding and pocket depth could be associated with smoking cessation. Such advice will prevent patients from any apprehension that may cause them to recommence smoking

Low-dose Warfarin Functions as an Immunomodulator to Prevent Cyclophosphamide-induced NOD Diabetes

Warfarin has been used as an anticoagulant for a long time. Recently, the pleiotropic effect of warfarin has been investigated. As low-dose warfarin has been reported to have anti-inflammatory effect through suppression of IL-6 secretion and inhibit the immune-associated signal between Tyro3 and its ligand, Gas6, the effect of low-dose warfarin on autoimmune diabetes in NOD mice was examined. To investigate the anti-inflammatory effect of warfarin, IL-6 secretion by splenocytes was examined in the presence of various concentrations of warfarin. Low concentration of warfarin inhibited IL-6 secretion. mRNA expression of Rse, one of the Tyro3 receptor family members, and Gas6 were analyzed in NOD mice. It was detected in islets, splenocytes and bone-marrow derived dendritic cells. 0.25 mg/l or 0.50 mg/l of warfarin was orally administered to NOD mice as a cyclophosphamide-induced diabetes model. Oral administration of warfarin at much lower doses than those clinically used as an anticoagulant significantly reduced the degree of insulitis and diabetes incidence in this model. We previously demonstrated that anti-FasL Ab-treatment led to complete prevention of autoimmune diabetes in NOD mice. As Fas/FasL signaling is reported to be essential for cyclophosphamide-induced diabetes model, we extracted RNA from lymphocytes of the inguinal lymph nodes of anti-FasL Ab-treated NOD mice and performed real-time PCR to determine expression of Rse gene. Interestingly, the expression of Rse gene related to the blockade of Fas/FasL signaling was reduced to less than half the level of untreated mice. In conclusion, low-dose warfarin is a potential immunomodulator which can prevent autoimmune diabetes. Type 1 diabetes is a chronic autoimmune disease caused by autoreactive T cells promoting the specific destruction of insulin-producing β cells of the pancreatic islets (1,6). Nonobese diabetic (NOD) mouse is an animal model of human autoimmune diabetes (19). In the NOD mouse, diabetes develops as the result of a chronic inflammation that starts with leukocytic infiltration of islets from 3-5 weeks of age and gradually exacerbates until hyperglycemia develops after 16 weeks of age in a high percentage of female mice. Warfarin has been widely used for a long time as an oral anticoagulant agent. In addition, Kater et al. reported the pleiotropic effect of low-dose warfarin related with inflammation, demonstrating that low-dose warfarin inhibited inflammatory signal transduction through suppression of TNF-α induced IL-6 secretion from murine macrophages (12)

Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation

Discovering dominant epitopes for T cells, particularly CD4+ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2-expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium (Be)-specific CD4+ T cells in the lung. We discovered lung resident CD4+ T cells that expressed a disease-specific public CDR3β T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligands 4 (CCL4) and 3 (CCL3). HLA-DP2-CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and 4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4+ T cells specific for the HLA-DP2-CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a vicious cycle of innate and adaptive immune activation

Beryllium-specific CD4\u3csup\u3e+\u3c/sup\u3e T cells induced by chemokine neoantigens perpetuate inflammation

Discovering dominant epitopes for T cells, particularly CD4+ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2–expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium-specific (Be-specific) CD4+ T cells in the lung. We discovered lung-resident CD4+ T cells that expressed a disease-specific public CDR3β T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligand 4 (CCL4) and CCL3. HLADP2–CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and CCL4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4+ T cells specific for the HLA-DP2–CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a cycle of innate and adaptive immune activation