Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

The role of zinc transporters in cadmium and manganese transport in mammalian cells

To understand the mechanism of cadmium accumulation, it is important to know the precise mechanisms of transport systems for other metals. Recently, utilization of genomics and metallomics has clarified the involvement of specific metal transporter(s) in cadmium uptake. Studies with metallothionein (MT)-null cadmium-resistant cells have revealed the involvement of the manganese/zinc transport system in cadmium uptake. Genomic studies of strain differences in sensitivity to cadmium-induced testicular hemorrhage revealed that a zinc transporter, Zrt-, Irt-related protein (ZIP) 8 encoded by slc39a8, is responsible for the strain difference. Ectopic expression of ZIP8 in various cells enhanced the uptake of cadmium, manganese, and zinc. ZIP8-transgenic mice showed high expression of ZIP8 in the vasculature of testis and apical membrane of proximal tubules in kidney, and exhibited enhanced cadmium accumulation and toxicity when treated with cadmium. The expression of ZIP8 was found to be down-regulated in MT-null cadmium-resistant cells, in which the uptake rates of both cadmium and manganese were decreased. These data suggest that ZIP8 plays an important role in the uptake of both cadmium and manganese in mammalian cells. The role of ZIP14 in the uptake of cadmium and manganese is also discussed.     

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.