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.     

Human cathelicidin antimicrobial peptide LL-37 promotes lymphangiogenesis in lymphatic endothelial cells through the ERK and Akt signaling pathways

Molecular Biology Reports - LL-37, the only member of the cathelicidin family of cationic antimicrobial peptides in humans has been shown to exhibit a wide variety of biological actions in addition...     

A continuation method for spatially discretized models with nonlocal interactions conserving size and shape of cells and lattices

Journal of Mathematical Biology - In this paper, we introduce a continuation method for the spatially discretized models, while conserving the size and shape of the cells and lattices. This...     

Fibroblasts-dependent invasion of podoplanin-positive cancer stem cells in squamous cell carcinoma

To clear whether podoplanin-positive cancer stem cells in squamous cell carcinoma have higher invasion activity during a fibroblasts-dependent invasion. A collagen gel invasion assay was performed using fluorescent ubiquitination-based cell cycle indicator-labeled A431 cells. The total number and number of invading cells in S/G2/M phase were counted using time-lapse imaging cocultured with fibroblasts. There was no significant difference between the number of invading podoplanin-positive and negative A431 cells when fibroblasts did not exist. On the contrary, the number of invading podoplanin-positive cells was significantly higher when fibroblasts existed. The frequency of cells in S/G2/M phase among invasion was no difference. Knockdown of podoplanin decreased the number of invaded A431 cells significantly when fibroblasts existed. Podoplanin-positive A431 cells display higher invasion activity when fibroblasts exist, suggesting that some biological functions of cancer stem cells might become evident only within the fibrous tumor microenvironment.     

Anisotropic intersubunit and inter-ring interactions revealed in the native bullet-shaped chaperonin complex from Thermus thermophilus

Background

The recent morphological studies on chaperonins have revealed that nearly equivalent amount of symmetric GroEL–(GroES)₂ (football-shaped) and asymmetric GroEL–GroES (bullet-shaped) complexes coexist during the chaperonin reaction cycle, which prompted us to reexamine the equatorial split observed for chaperonin from Thermus thermophilus by implementing semi-empirical molecular orbital (MO) calculations, since it is now believed that the symmetric formation is a precursor to the equatorial split.

Methods

Semi-empirical MO calculations were employed to investigate the intersubunit interactions within the bullet-shaped T. thermophilus chaperonin capturing the substrate of folding intermediates. Interaction energies between each cis-GroEL subunit and closely related remaining subunits in cis-GroEL ring, or in trans-GroEL ring across the equatorial plane, and further, interaction energies between each GroES subunit and adjacent subunits in the same GroES ring and in cis-GroEL ring were simulated.

Results

Anisotropic intensities and energy distribution of the subunits were revealed by the calculations, which are consistent with two conformations of the subunits forming cis-GroEL ring as revealed by X-ray crystal structure, and with an anisotropic critical binding site on cis-GroEL ring for chaperonin functioning.

Conclusions

This is the first application of semi-empirical MO calculations to the macromolecular complex of the native bullet-shaped chaperonin (GroEL–GroES–ADP homolog) from T. thermophilus.

General significance

The results also appear to support the occurrence of the equatorial split for T. thermophilus chaperonin observed via electron microscopy, but has not yet been fully observed for Escherichia coli GroEL–GroES system.     

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45⁺ cells, especially Gr-1^high CD11b⁺ MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.     

Neurite Outgrowth of PC12 Cells by 4′-O-β-d-Glucopyranosyl-3′,4-Dimethoxychalcone from Brassica rapa L. ‘hidabeni’ was Enhanced by Pretreatment with p38MAPK Inhibitor

The cellular effects of eleven compounds including chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ and their synthetic derivatives were studied in rat pheochromocytoma PC12 cells. Of the compounds tested, 4′-O-β-d-glucopyranosyl-3′,4-dimethoxychalcone (A2) significantly increased the levels of the phosphorylated forms of extracellular signal-regulated kinases 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38MAPK), and stress-activated protein kinases/Jun amino-terminal kinases (JNK/SAPK), but it did not affect Akt. Nerve growth factor (NGF), a well-known neurotrophic factor, increased the levels of phosphorylated ERK1/2, JNK/SAPK, and Akt but not p38MAPK, which may mediate marked neurite outgrowth. Signals evoked by A2 shared common characteristics with those induced by NGF; therefore, we evaluated the neuritogenic activity of A2 and found it induced only weak neurite outgrowth. However, this effect was enhanced by pre-treatment with a p38MAPK inhibitor, suggesting that the phosphorylation of p38MAPK down-regulated neurite outgrowth. From the results of this study, it was found that A2 in combination with a p38MAPK inhibitor can induce NGF-like effects. Hence, a combination of chalcone glycosides containing A2 and a p38MAPK inhibitor increases the likelihood that chalcone glycosides could be put to practical use in the form of drugs or alternative medicines to maintain neural health.     

Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial‐resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P = 0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm‐made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm‐made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to > 512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial‐resistant LAB in imported and Japanese farm‐made cheeses on the Japanese market, but not in Japanese commercial cheeses.     

Molecular Dynamics Calculations of Wild Type vs. Mutant Protein C: Relationship Between Binding Affinity to Endothelial Cell Protein C Receptor and Hereditary Disease

Abstract

Molecular dynamics simulations of the protein C γ-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 → Lys) in the protein C Gla domain. Our results suggest that the Gla 25 → Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca²⁺ ion.     

Root orientation can affect detection accuracy of ground-penetrating radar

Aim

Ground-penetrating radar (GPR) has been applied to detect coarse tree roots. The horizontal angle of a root crossing a scanning line is a factor that affects both root detection and waveform parameter values. The purpose of this study was to quantitatively evaluate the influence of root orientation (x, degree) on two major waveform parameters, amplitude area (A, dB × ns) and time interval between zero crossings (T, ns).

Methods

We scanned four diameter classes of dowels in a sandy bed as simulated roots using a 900 MHz antenna from multiple angles to clarify the relationships between the parameters and x.

Results

Angle x strongly affected reflection images and A values. The variation in A(x) fitted a sinusoidal waveform, whereas T was independent of x. The value of A scanning at 90° was estimated by A values of arbitrary x in two orthogonal transects. The sum of T in all reflected waveforms showed a significant linear correlation with dowel diameter.

Conclusions

We clarified that root orientation dramatically affected root detection and A values. The sum of T of all reflected waveforms was a suitable parameter for estimating root diameter. Applying grid transects can overcome the effects of root orientation.