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.     

Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-β administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-β alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-β and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-β, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.     

High-efficiency protein delivery into transfection-recalcitrant cell types

Intracellular delivery of functional proteins is of great interest for basic biological research as well as for clinical applications. Transfection is the most commonly used method, however, it is not applicable to large-scale manipulation and inefficient in important cell types implicated in biomedical applications, such as epithelial, immune and pluripotent stem cells. In this study, we explored a bacterial type III secretion system (Bac-T3SS)-mediated proteofection method to overcome these limitations. An attenuated Pseudomonas aeruginosa vector was constructed, which has features of low toxicity, high T3SS activity, and self-limiting growth. Compared to the method of transfection, the Bac-T3SS showed significantly higher efficiencies of Cre recombinase translocation and target site recombination for hard-to-transfect human cell lines. Furthermore, through the delivery of β-lactamase in live animals, we demonstrated the feasibility and biosafety of in vivo application of the Bac-T3SS. This study provided an efficient and low-cost proteofection strategy for laboratory use as well as for application in large-scale cell manipulations.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps

VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1–3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function.     

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.     

Ninety-year-, but not single, application of phosphorus fertilizer has a major impact on arbuscular mycorrhizal fungal communities

Background and aims

Arbuscular mycorrhizal (AM) fungi play a significant role in P nutrition of crops in agriculture, but P accumulation in the soil, e.g., application of P-fertilizer, generally reduces AM fungal colonization. The impact of long-term application of chemical fertilizer on AM fungal communities was investigated with respect to the time scale.

Methods

Soils were collected from four plots with different fertilizer management in the long-term experimental field established in 1914. Lotus japonicus was grown in the soils in a greenhouse, while Glycine max was grown in the plots in the field. DNA was extracted from their roots, and the diversity and community compositions were analyzed based on occurrence of the AM fungal phylotypes defined by sequence similarity in the LSU rDNA.

Results

The 90-year-application of N and K in the absence of P increased AM fungal diversity and resulted in formation of a distinctive fungal community compared with those in the other treatments. This effect was not cancelled by single application of P. Whereas the impact of balanced application of N, P, and K was ambiguous.

Conclusion

These observations suggest that the presence/absence of P-fertilizer has a major impact on AM fungal communities, but the action may appear only on a long time scale.     

Crystal Structures of β-1,4-Galactosyltransferase 7 Enzyme Reveal Conformational Changes and Substrate Binding

The β-1,4-galactosyltransferase 7 (β4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human β4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type β4GalT7 and D211N β4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N β4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an S_N2 type catalytic mechanism for the β4GalT7 enzyme.     

Association analysis of the HLA-C gene in Japanese alopecia areata

Alopecia areata (AA) is an organ-specific and cell-mediated autoimmune disease involving hair loss, but its pathogenesis remains poorly understood. Many autoimmune diseases are genetically associated with alleles of the human leukocyte antigen (HLA) genes within the major histocompatibility complex. Associations between AA and HLA genes were previously observed in some different ethnic groups. However, the results were inconsistent, and a primary susceptibility HLA gene and/or region has not yet been assigned for AA. The aim of this study was to evaluate whether an allele of the HLA-C locus, HLA-C*07:04, which was strongly associated with AA in Chinese Hans, could be replicated in the Japanese population. The HLA-C locus was genotyped by the SSO method using 156 AA patients and 560 healthy controls. As a consequence, among the 17 alleles detected, only two alleles, C*04:01 (OR?=?2.25, CI 95 %?=?1.35–3.75, P?=?1.84E-03) and C*15:02 (OR?=?2.52, CI 95 %?=?1.37–4.64, P?=?2.90E-03), were significantly associated with AA after Bonferroni correction. Further, the stratification analysis suggested that C*04:01, C*07:02, and C*15:02 represented different AA genetic risk factors in each sub-phenotype.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).