Platelets Promote Tumor Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus–CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus–CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet–tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.     

Inhibitory synaptic transmission tuned by Ca2+ and glutamate through the control of GABAAR lateral diffusion dynamics

The GABAergic synapses, a primary inhibitory synapse in the mammalian brain, is important for the normal development of brain circuits, and for the regulation of the excitation-inhibition balance critical for brain function from the developmental stage throughout life. However, the molecular mechanism underlying the formation, maintenance, and modulation of GABAergic synapses is less understood compared to that of excitatory synapses. Quantum dot-single particle tracking (QD-SPT), a super-resolution imaging technique that enables the analysis of membrane molecule dynamics at single-molecule resolution, is a powerful tool to analyze the behavior of proteins and lipids on the plasma membrane. In this review, we summarize the recent application of QD-SPT in understanding of GABAergic synaptic transmission. Here we introduce QD-SPT experiments that provide further insights into the molecular mechanism supporting GABAergic synapses. QD-SPT studies revealed that glutamate and Ca²⁺ signaling is involved in (a) the maintenance of GABAergic synapses, (b) GABAergic long-term depression, and GABAergic long-term potentiation, by specifically activating signaling pathways unique to each phenomenon. We also introduce a novel Ca²⁺ imaging technique to describe the diversity of Ca²⁺ signals that may activate the downstream signaling pathways that induce specific biological output.     

Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.     

Effects of Sphingomyelin Headgroup Size on Interactions with Ceramide

Sphingomyelins (SMs) and ceramides are known to interact favorably in bilayer membranes. Because ceramide lacks a headgroup that could shield its hydrophobic body from unfavorable interactions with water, accommodation of ceramide under the larger phosphocholine headgroup of SM could contribute to their favorable interactions. To elucidate the role of SM headgroup for SM/ceramide interactions, we explored the effects of reducing the size of the phosphocholine headgroup (removing one, two, or three methyls on the choline moiety, or the choline moiety itself). Using differential scanning calorimetry and fluorescence spectroscopy, we found that the size of the SM headgroup had no marked effect on the thermal stability of ordered domains formed by SM analog/palmitoyl ceramide (PCer) interactions. In more complex bilayers composed of a fluid glycerophospholipid, SM analog, and PCer, the thermal stability and molecular order of the laterally segregated gel domains were roughly identical despite variation in SM headgroup size. We suggest that that the association between PCer and SM analogs was stabilized by ceramide’s aversion for disordered phospholipids, by interfacial hydrogen bonding between PCer and the SM analogs, and by attractive van der Waals’ forces between saturated chains of PCer and SM analogs.     

Hyaluronan synthesis in cultured tobacco cells (BY‐2) expressing a chlorovirus enzyme: Cytological studies

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured‐cells (BY‐2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY‐2 cells harboring a vSPO‐cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N‐terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Biotechnol. Bioeng. 2013; 110: 1174–1179. © 2012 Wiley Periodicals, Inc.     

Studies on Leaf Oils of Citrus Species

Leaf oil samples of four Japanese citrus species were analysed by gas chromatography to determine the detailed composition of each leaf oil. The following components were identified: α-pinene, α-thujene, camphene, β-pinene, sabinene, β-myrcene, α-terpinene, limonene, β-phellandrene, trans-2-hexen-1-al, γ-terpinene, p-cymene, terpinolene, cis-2-penten-1-ol, n-hexyl alcohol, cis-3-hexen-1-ol, trans-2-hexen-1-ol, p-α-dimethylstyrene linalool, linalyl acetate, β-elemene, terpinen-4-ol, caryophyllene, humulene, α-terpineol, neryl acetate, geranyl acetate, β-selinene, geraniol and thymol. Most components were contained in common in leaf oils of the four citrus species, but relative contents of some of the components; such as γ-terpinene, linalyl acetate, and thymol differed from species to species. For example, γ-terpinene was the major component (33.8%) of Hassaku, whereas it was only a minor component in Daidai. Daidai is characterized by a very high content of linalyl acetate (35%) which is only a trace in the other three species. Kishu-mikan is characterized by a high content of thymol (15%).     

5,7-Dodecadien-1-ol: Candidate for the Sex Pheromone of the Pine Moth

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from Spinacia oleracea. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 86,000 by Ultrogel AcA 34 gel filtration. In the oxidized form, the enzyme had absorption maxima at 278, 388 (Soret band), 573 (α band) and 690 nm, indicating that siroheme is directly involved in the catalysis of nitrite reduction. This absorption spectrum was modified by sulfite, hydroxylamine and cyanide. The enzyme exhibited electron paramagnetic resonance signals with g values of 6.9 and 5.2, which are characteristic of a high spin Fe³⁺ -siroheme in the molecule. These signals disappeared upon the addition of dithionite or nitrite. This isolated enzyme also contained four moles of labile sulfide and 7 g-atoms of iron per 86,000 g of protein.     

Structure-Activity Study of S-n-Butyl S′-p-teri-Butylbenzyl N-3-Pyridyldithiocarbonimidate (S-1358, Denmert®) and Its Derivatives

Fungicidal activities of S-alkyl S′-p-substituted benzyl N-3-pyridyldithiocarbonimidates toward Coniothyrium diplodiella, Scleotinia sclerotiorum (on agar medium) and Sphaerotheca fuliginea (on pot test) were shown. The activities varied with the change of the S-alkyl group and were maximized at certain numbers of carbon atoms. A different pattern was observed in the activity toward S. fuliginea when the carbon number was 8 or more. Bulkiness of the S-alkyl group also appeared to influence the activities. The steric factor of the p-alkyl substituents mainly influenced the activity toward S. fuliginea up to the tert-butyl analog. The activities toward C. diplodiella and S. sclerotiorum increased with the increase in the bulkiness and the hydrophylicity of the p-substituent. Rm values were determined on reversed phase TLC and the structure-activity correlations were analyzed against hydrophobic (Rm), electronic (σ) and steric (Es) factors on 34 compounds.