Comparison of Thermogenic Sympathetic Response to Food Intake between Obese and Non‐obese Young Women

Objective: Sympathetic nervous system abnormality in humans is still a matter of debate. The present study was designed to examine diet‐induced autonomic nervous system activity and metabolic change in obese and non‐obese young women. Research Methods and Procedures: Sixteen age‐ and height‐matched obese and non‐obese young women participated in this study. Sympathovagal activities were assessed by means of our newly developed spectral analysis procedure of heart‐rate variability during the resting condition and after mixed‐food ingestion (480 kcal). Energy expenditure was also measured under these two conditions. Results: There was no significant difference in any of the parameters of the heart‐rate variability between the obese group and control group during the resting condition. In the control group, both absolute values (221.5 ± 54.5 vs. 363.8 ± 43.7 ms², p < 0.05) and relative values (0.23 ± 0.03 to 0.36 ± 0.02, p < 0.05) of a very‐low‐frequency component and global sympathetic nervous system index (1.46 ± 0.19 vs. 3.26 ± 0.61, p < 0.05) were significantly increased after mixed‐food ingestion compared with the values obtained after resting condition. However, no such sympathetic response was found in the obese group. Energy expenditure increased in the two groups after the meal, but the magnitude of the increase above the preprandial resting condition was significantly greater in the control group than in the obese group (11.2 ± 2.3 vs. 6.7 ± 0.8%, p < 0.05). Discussion: Our data suggest that despite identical sympathovagal activities at the resting condition, obese young women may possess a reduced sympathetic response to physiological perturbation such as mixed food intake, which might be related to lowered capacity of thermogenesis and the state of obesity.     

Association between IFNA genotype and the risk of sarcoidosis

Sarcoidosis is known to be a systemic granulomatous disorder characterized by a cell-mediated Th1-type inflammatory response. To identify a key genetic factor in the pathogenesis of sarcoidosis, we investigated single nucleotide polymorphisms within 10 candidate genes involved in type 1 immune process (IFNA17, IFNB, IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1, IL12RB2, ETA-1, and NRAMP1) in an association-based study of 102 Japanese patients with sarcoidosis, 114 with tuberculosis, and 110 control subjects. After correction for multiple testing, an IFNA17 polymorphism (551TG) was found to be associated with susceptibility to sarcoidosis (odds ratio 3.27 [95% CI: 1.44–7.46], P=0.004, P_c=0.04), but not to tuberculosis. We observed no significant associations with the other polymorphisms of the Th1-related genes. We further typed another IFNA polymorphism (IFNA10 60TA) and confirmed two major haplotypes of the IFNA gene, viz., allele 1: IFNA10 [60T]-IFNA17 [551T] and allele 2: IFNA10 [60A]-IFNA17 [551G], in the Japanese population. In healthy subjects, IFNA allele 2, which is over-represented in patients with sarcoidosis, was significantly associated with increased IFN- and IL-12p70 production induced by Sendai virus in vitro. This study suggests that possession of the IFNA allele with higher levels of IFN- significantly increases the risk of sarcoidosis.M. Akahoshi and M. Ishihara contributed equally to this work     

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.     

Rapid Communication: GM3 Regulates Protein Kinase Systems in Cultured Brain Microvascular Endothelial Cells

Abstract: The barrier function of endothelial cells is known to be positively regulated by protein kinase A (PKA) and negatively regulated by protein kinase C (PKC). We found that exogenously administered GM3(NeuAc) promoted PKA activity in cultured brain microvascular endothelial cells (BMECs). Other glycolipids, including GM1, sulfoglucuronyl paragloboside, and GM3(NeuGc), did not have any effect on the PKA activity of BMECs. PC12 cells did not respond to exogenously applied GM3(NeuAc). GM3(NeuAc) also suppressed the PKC activity of BMECs. Thus, GM3(NeuAc) may function as a modulator of blood-brain barrier function via the two different kinase systems.     

The Human Cytomegalovirus UL76 Gene Regulates the Level of Expression of the UL77 Gene

Background

Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5′ mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein.

Methodology/Principal Findings

To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth.

Conclusions/Significance

While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells.     

Mutation analysis of 2009 pandemic influenza A(H1N1) viruses collected in Japan during the peak phase of the pandemic

Background

Pandemic influenza A(H1N1) virus infection quickly circulated worldwide in 2009. In Japan, the first case was reported in May 2009, one month after its outbreak in Mexico. Thereafter, A(H1N1) infection spread widely throughout the country. It is of great importance to profile and understand the situation regarding viral mutations and their circulation in Japan to accumulate a knowledge base and to prepare clinical response platforms before a second pandemic (pdm) wave emerges.

Methodology

A total of 253 swab samples were collected from patients with influenza-like illness in the Osaka, Tokyo, and Chiba areas both in May 2009 and between October 2009 and January 2010. We analyzed partial sequences of the hemagglutinin (HA) and neuraminidase (NA) genes of the 2009 pdm influenza virus in the collected clinical samples. By phylogenetic analysis, we identified major variants of the 2009 pdm influenza virus and critical mutations associated with severe cases, including drug-resistance mutations.

Results and Conclusions

Our sequence analysis has revealed that both HA-S220T and NA-N248D are major non-synonymous mutations that clearly discriminate the 2009 pdm influenza viruses identified in the very early phase (May 2009) from those found in the peak phase (October 2009 to January 2010) in Japan. By phylogenetic analysis, we found 14 micro-clades within the viruses collected during the peak phase. Among them, 12 were new micro-clades, while two were previously reported. Oseltamivir resistance-related mutations, i.e., NA-H275Y and NA-N295S, were also detected in sporadic cases in Osaka and Tokyo.     

Increase of nitrosative stress in patients with eosinophilic pneumonia

Background

Exhaled nitric oxide (NO) production is increased in asthma and reflects the degree of airway inflammation. The alveolar NO concentration (Calv) in interstitial pneumonia is reported to be increased. However, it remains unknown whether NO production is increased and nitrosative stress occurs in eosinophilic pneumonia (EP). We hypothesized that nitrosative stress markers including Calv, inducible type of NO synthase (iNOS), and 3-nitrotyrosine (3-NT), are upregulated in EP.

Methods

Exhaled NO including fractional exhaled NO (FE_NO) and Calv was measured in ten healthy subjects, 13 patients with idiopathic pulmonary fibrosis (IPF), and 13 patients with EP. iNOS expression and 3-NT formation were assessed by immunocytochemistory in BALf cells. The exhaled NO, lung function, and systemic inflammatory markers of the EP patients were investigated after corticosteroid treatment for 4 weeks.

Results

The Calv levels in the EP group (14.4 ± 2.0 ppb) were significantly higher than those in the healthy subjects (5.1 ± 0.6 ppb, p < 0.01) and the IPF groups (6.3 ± 0.6 ppb, p < 0.01) as well as the FE_NO and the corrected Calv levels (all p < 0.01). More iNOS and 3-NT positive cells were observed in the EP group compared to the healthy subject and IPF patient. The Calv levels had significant positive correlations with both iNOS (r = 0.858, p < 0.05) and 3-NT positive cells (r = 0.924, p < 0.01). Corticosteroid treatment significantly reduced both the FE_NO (p < 0.05) and the Calv levels (p < 0.01). The magnitude of reduction in the Calv levels had a significant positive correlation with the peripheral blood eosinophil counts (r = 0.802, p < 0.05).

Conclusions

These results suggested that excessive nitrosative stress occurred in EP and that Calv could be a marker of the disease activity.     

The roles of cadherins and nectins in interneuronal synapse formation

Cadherins are Ca(2+)-dependent intercellular adhesion molecules (CAMs) and they play key roles in the intercellular junctions of a wide variety of cells, including interneuronal synapses. Nectins are Ca(2+)-independent immunoglobulin-like CAMs and they are also involved in the organization of various types of intercellular junctions, including interneuronal synapses, either in cooperation with or independently of cadherins. Intercellular adhesion through nectins induces activation of Cdc42 and Rac small G proteins, leading to a reorganization of the actin cytoskeleton, gene expression, and cell polarization.     

Heterothallic life cycle in the white root rot fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

To prepare homologous DNA fragments as restriction fragment length polymorphism (RFLP) markers, the genes encoding phenol oxidase, chitinase, and xylanase were amplified from genomic DNA of Rosellinia necatrix strains. RFLP analysis using the amplified DNA fragments as probe was carried out, with segregation of the markers among two sets of F₁ progenies isolated from an independent perithecium. RFLP was frequently found using rpo1 as the RFLP marker among strains of R. necatrix, which was isolated from single ascospores and the circumference of the perithecium. In each set, RFLPs of some F₁ progenies were different from that of the parent strain. Random amplified polymorphic DNA (RAPD) also revealed that several strains, which were of different genotypes from the parent strain, were contained in the single ascospore culture isolated from the same perithecium. From these results, it is suggested that another strain, which was genetically different, was required for mating and development of the ascus in R. necatrix. Therefore, the life cycle in R. necatrix was presumed to be heterothallism. This is the first report about a heterothallic life cycle in R. necatrix.