Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

Cetuximab is a chimeric mouse–human monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). However, EGFR expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer (CRC) patients treated with cetuximab. Therefore, we evaluated the correlation between EGFR levels detected by cetuximab and drug sensitivities of CRC cell lines (Caco-2, WiDR, SW480, and HCT116) and the A431 epidermoid carcinoma cell line. We used flow cytometry (FCM) to detect EGFR-binding of biotinylated cetuximab on the cell surface. Subcloned cell lines showing the highest and lowest EGFR expression levels were chosen for further study. Cytotoxic assays were used to determine differential responses to cetuximab. Xenograft models treated with cetuximab intraperitoneally to assess sensitivity to cetuximab. Strong responses to cetuximab were specifically exhibited by subcloned cells with high EGFR expression levels. Furthermore, cetuximab inhibited the growth of tumors in xenograft models with high or low EGFR expression levels by 35% and 10%–20%, respectively. We conclude that detection of EGFR expression by cetuximab promises to provide a novel, sensitive, and specific method for predicting the sensitivity of CRC to cetuximab.     

CLE2 regulates light-dependent carbohydrate metabolism in Arabidopsis shoots

Plant Molecular Biology - This study focused on the role of CLE1-CLE7 peptides as environmental mediators and indicated that root-induced CLE2 functions systemically in light-dependent carbohydrate...     

Spatial covariation of fish population vital rates in a stream network

Animal populations are spatially structured in heterogeneous landscapes, in which local patches with differing vital rates are connected by dispersal of individuals to varying degrees. Although there is evidence that vital rates differ among local populations, much less is understood about how vital rates covary among local patches in spatially heterogeneous landscapes. In this study, we conducted a nine-year annual mark–recapture survey to characterize spatial covariation of survival and growth for two Japanese native salmonids, white-spotted charr Salvelinus leucomaenis japonicus and red-spotted masu salmon Oncorhynchus masou ishikawae, in a headwater stream network composed of distinctly different tributary and mainstem habitats. Spatial structure of survival and growth differed by species and age class, but results provided support for negative covariation between vital rates, where survival was higher in the tributary habitat but growth was higher in the mainstem habitat. Thus, neither habitat was apparently more important than the other, and local habitats with complementary vital rates may make this spatially structured population less vulnerable to environmental change (i.e. portfolio effect). Despite the spatial structure of vital rates and possibilities that fish can exploit spatially distributed resources, movement of fish was limited due partly to a series of low-head dams that prevented upstream movement of fish in the study area. This study shows that spatial structure of vital rates can be complex and depend on species and age class, and this knowledge is likely paramount to elucidating dynamics of spatially structured populations.     

Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene,Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β₂‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

FCGR3A-158 polymorphism influences the biological response to infliximab in Crohn’s disease through affecting the ADCC activity

An association between FCGR3A-158 V/F polymorphism and biological responses to infliximab has been reported in Crohn’s disease (CD) in Western countries. However, little is known about the mechanism by which gene polymorphism affects the responses to infliximab. The aims of this study were to confirm the association in Japanese CD patients and to reveal the effect of gene polymorphism on biological responses to infliximab. Japanese CD patients were examined retrospectively at weeks 8 and 30. Clinical and biological responses were assessed by the Crohn’s disease activity index and C-reactive protein levels, respectively. The infliximab-binding affinity of natural killer (NK) cells from FCGR3A-158 V/V, V/F and F/F donors was examined. Infliximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activities were also determined using transmembrane TNF-α-expressing Jurkat T cells as target cells and peripheral blood mononuclear cells (PBMCs) from V/V, V/F and F/F donors as effector cells. Biological responses at week 8 were statistically higher in V/V patients, whereas no significant differences were observed in either clinical responses at weeks 8 and 30 or biological responses at week 30 among the three genotypes. NK cells and PBMCs from V/V patients also showed higher infliximab-binding affinity and infliximab-mediated ADCC activity, respectively. Our results suggest that FCGR3A-158 polymorphism is a predicting factor of biological responses to infliximab in the early phases. FCGR3A-158 polymorphism was also found to affect the infliximab-binding affinity of NK cells and infliximab-mediated ADCC activity in vitro, suggesting that an effect on ADCC activity influences biological responses to infliximab in CD patients.     

Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

Background

A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function.

Methods

Differentiated 3T3-L1 adipocytes were incubated at 5% O₂ for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC).

Results

HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01).

Conclusion

We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via decreased glucose uptake and lipogenic protein expression and increased basal lipolysis. Such an hypoxia-induced decrease in lipogenesis may be an attractive therapeutic target against lipid-associated metabolic diseases.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

In vitro mycorrhization and acclimatization of Amanita caesareoides and its relatives on Pinus densiflora

Amanita caesareoides is a sister species of Amanita caesarea, also known as Caesar’s mushroom and one of the most desirable edible mycorrhizal mushrooms. However, cultivation of Caesar’s mushrooms has not yet been successful due to the difficulties involved in establishing pure cultures. In this study, we established pure cultures of four Asian Caesar’s mushroom species, i.e., A. caesareoides, Amanita javanica, Amanita esculenta, and Amanita similis, which were identified by sequence analysis of their rDNA internal transcribed spacer (ITS) region. Five selected isolates in A. caesareoides, A. javanica, and A. esculenta were tested for ectomycorrhizal syntheses with axenic Pinus densiflora seedlings in vitro. Ectomycorrhizal tips of each fungal isolate tested were observed on pine lateral roots within 5 months of inoculation. Seventeen pine seedlings that formed ectomycorrhizas in vitro with these three Amanita species were acclimatized under non-sterile conditions. Seven months following acclimatization, ectomycorrhizal colonization by A. caesareoides was observed on newly grown root tips, which was confirmed by polymerase chain reaction restriction fragment length polymorphism analysis of the fungal rDNA ITS region. Two other Amanita species also survived during ectomycorrhizal acclimatization. These results suggest that the cultivation of A. caesareoides and its relatives can be attempted through mycorrhizal synthesis using P. densiflora as a host. This is the first report of in vitro mycorrhization of Asian Caesar’s mushrooms and their acclimatization under non-sterile conditions.