Antigen-specific augmentation of delayed-type hypersensitivity by immune serum factor in mice: augmentation of anti-tumor cytostatic activity

A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DFR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effectors, respectively. The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. However, a sufficient number of macrophages were always required as the final effectors in their functional expression. These primary effectors were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seems to exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responsible for cytostatic anti-tumor immunity.     

Antiangiogenic Tumor Therapy by DNA Vaccine Inducing Aquaporin-1-Specific CTL Based on Ubiquitin-Proteasome System in Mice

Aquaporin-1 (AQP-1) is a water channel protein highly expressed in the vascular endothelial cells of proliferating tissues including malignant cancers. Given that in APC ubiquitinated peptides are effectively introduced into proteasomes from which CD8 epitopes are excised, we fused ubiquitin with AQP-1 (pUB-AQP-1) to produce a DNA vaccine. In C57BL/6J mice immunized with pUB-AQP-1, the growth of B16F10 melanoma was profoundly inhibited. The antitumor effect of the pUB-AQP-1 DNA vaccine was largely mediated by CD8 T cells, which secrete IFN-γ, perforin, and granzyme-B in the presence of APCs transfected with pUB-AQP-1. AQP-1-specific CD8 T cells possessed cytotoxic activity both in vivo and in vitro. After tumor challenge, the microvessel density decreased and the ratio of total blood vessel area to tumor area was significantly reduced as compared with control mice, resulting in a dramatic suppression of tumor growth. The immunization effect was completely abrogated in immunoproteasome-deficient mice. Strikingly this pUB-AQP-1 DNA vaccine was also effective against Colon 26 colon tumors (BALB/c) and MBT/2 bladder tumors (C3H/HeN). Thus, this ubiquitin-conjugated DNA immunization-targeting tumor vasculature is a valid and promising antitumor therapy. This vaccine works across the barriers of tumor species and MHC class I differences in host mice.     

Heterologous expression and functional characterization of a novel chitinase from the chitinolytic bacterium Chitiniphilus shinanonensis

Chitiniphilus shinanonensis strain SAY3(T) is a chitinolytic bacterium isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. Fifteen genes encoding putative chitinolytic enzymes (chiA-chiO) have been isolated from this bacterium. Five of these constitute a single operon (chiCDEFG). The open reading frames of chiC, chiD, chiE, and chiG show sequence similarity to family 18 chitinases, while chiF encodes a polypeptide with two chitin-binding domains but no catalytic domain. Each of the five genes was successfully expressed in Escherichia coli, and the resulting recombinant proteins were characterized. Four of the recombinant proteins (ChiC, ChiD, ChiE, and ChiG) exhibited endo-type chitinase activity toward chitinous substrates, while ChiF showed no chitinolytic activity. In contrast to most endo-type chitinases, which mainly produce a dimer of N-acetyl-D-glucosamine (GlcNAc) as final product, ChiG completely split the GlcNAc dimer into GlcNAc monomers, indicating that it is a novel chitinase.     

A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling

In RNA site-directed spin labeling (SDSL) studies, structural and dynamic information at the individual RNA nucleotide level is derived from the observed electron paramagnetic resonance spectrum of a covalently attached nitroxide. A systematic approach for RNA SDSL is to establish a library that categorizes observed spectral lineshapes based on known RNA structures, thus enabling lineshape-based structure identification at any RNA site. To establish the first RNA SDSL library, selective secondary structure elements have been systematically engineered into a model RNA. Nitroxide lineshapes reporting features specific to each element were obtained utilizing a new avidin-tethering scheme for suppressing spectral effects due to uniform RNA tumbling. The data demonstrated two key features required for a SDSL library with a predicting power: (i) spectral divergence--distinctive lineshape for different elements; and (ii) spectral convergence--similar lineshape for the same element in different contexts. This sets the foundation for further RNA SDSL library development.     

Sec22b Is a Negative Regulator of Phagocytosis in Macrophages

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.     

Relationships Between Slc1a5 and Osteoclastogenesis

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5^−/− mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5^+/+ and Slc1a5^−/− bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5^−/− bone marrow cells was reduced to 70% of that of cells from Slc1a5^+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5^+/+ or Slc1a5^−/− mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5^−/− mice was severely impaired compared with those from Slc1a5^+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5^−/− osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Osteoclasts are giant multinucleated cells of hematopoietic origin that are responsible for bone resorption. The differentiation of osteoclasts can be induced by treating bone marrow macrophages with RANKL.² After stimulation, bone marrow macrophages mature and then fuse to become multinucleated osteoclasts. The processes of osteoclastogenesis and bone resorption are known to be energy-demanding,⁸ but little is known about the amino acid requirements of osteoclasts. In this study, we investigated the role of glutamine in osteoclastogenesis. Glutamine was selected for this work because it provides an excellent example of amino acid metabolism.Although glutamine acts as an essential amino acid in some specific physiologic situations, it is classified as a nonessential amino acid.⁵ The need for the biosynthesis and metabolism of amino acids is significantly increased in cells with high rates of proliferation, such as functionally active cells and cancer cells. The activity of amino acid synthetases such as glutamine synthetase is increased in these cells. In addition, glutamine transporters on the plasma membrane are important, because they mediate glutamine uptake to meet the intracellular glutamine demand. The transporter Slc1a5, also known as ASCT2, is particularly important for glutaminolysis and mTOR signaling.^14,16Glutamine concentrations in tissue and blood are regulated by the activities of glutamine synthetase and glutaminase. ­Endogenous synthesis cannot meet the cell’s demands for glutamine in conditions including cancer, infections, and intense physical exercise. Glutamine is released into the blood from the lungs, adipocytes, and skeletal muscles and is transported into the cytoplasm via glutamine acid transporter molecules on the cell membrane. Glutamine is required for the growth of cancer cells; upregulation of the expression of the proteins involved in glutamine transport has been observed in tumor cells.⁴ Slc1a5 (ASCT2) is a small neutral amino acid exchanger that is overexpressed in many cancers and is the most well-described glutamine transporter in cancer cells.⁹ However, previous studies^1,10,22,23 have reported that silencing, deletion, and amino-acid analog substitution of Slc1a5 in cancer cells generated different results for mTORC1 signaling, proliferation, and cell migration.^{1,3,4,10,22,23} Additional work^3,4 has shown that Slc1a5 is indispensable for tumor growth and mTORC1 signaling. Slc1a5 is important in accumulating nonessential amino acids to quickly restore amino acid composition during imbalanced amino acid usage,⁴ whereas Slc38a1 (SNAT1) and Slc38a2 (SNAT2) mediate the net import of glutamine.In bone homeostasis, glutamine is a critical regulator of energy for protein and nucleic acid synthesis via the tricarboxylic acid cycle. Active glutamine metabolism stimulates the proliferation and differentiation of osteoblasts, chondrocytes, and osteoclasts. The enzyme glutaminase deaminates glutamine to form glutamate. Glutaminase deficiency in osteoblasts and chondrocytes leads to reduced osteoblast formation and decreased bone mass, resulting in potentially dangerous conditions, such as osteoporosis.²⁴ In osteoclasts, glutamine is an important source of fuel for protein and nucleic acid biosynthesis. Therefore, Slc1a5 deficiency in mice may influence bone homeostasis, including osteoclastogenesis. We therefore created Slc1a5-deficient mice to investigate the contribution of Slc1a5 to the development and functional properties of osteoclasts.     

Novel factor rescues ribosomes trapped on non-stop mRNAs

Ribosomes are trapped at the 3′ ends of mRNAs that lack a natural stop codon. In bacteria, a reaction called trans-translation recycles ribosomes entrapped at such ‘non-stop’ mRNAs. The main player in trans-translation is tmRNA (SsrA-RNA), a bi-functional RNA that acts as both a tRNA and an mRNA. In the trans-translation reaction, alanine-charged tmRNA loads at the ribosomal A-site and translation shifts to the resume codon in tmRNA. Translation of tmRNA stops at a natural stop codon at the end of the small reading frame of tmRNA. In this way, the reaction simultaneously adds a peptide tag to the end of the nascent, incomplete polypeptide and recycles the stalled ribosomes. The peptide tag is recognized by cellular proteases that rapidly degrade the incomplete, potentially harmful polypeptides. The trans-translation reaction is not essential in most bacteria, raising the possibility that ribosomes stalled at non-stop mRNAs can be rescued by alternative routes. In this issue of Molecular Microbiology, Chadani et al. show that a novel translation factor, ArfA, can recycle a ribosome trapped at the 3′ end of a non-stop mRNA in the absence of trans-translation. AfrA is essential in the absence of tmRNA, showing that the two systems work in parallel to resolve stalled ribosomes.