Biochemical characterization of the <Emphasis Type="Italic">Arabidopsis</Emphasis> KS-type dehydrin protein,whose gene expression is constitutively abundant rather than stress dependent

Dehydrins are known as plant stress-responsive genes. Arabidopsis thaliana has 10 dehydrin genes. Among them, one of the highly expressed genes is a KS-type dehydrin (At1g54410). However, the gene product, which is a histidine-rich dehydrin whose molecular mass is 11 kDa (AtHIRD11), has not been studied. Thus, we report the biochemical characterization of the AtHIRD11 protein. Although the AtHIRD11 protein was detected in all organs of Arabidopsis, the bolting stem and the flower showed higher accumulation than the other organs, with the AtHIRD11 protein detected in the cambial zone of the stem vasculature. Most of the AtHIRD11 protein was found to be a bound form. The bound AtHIRD11 was solubilized by 1 M NaCl solution. The extracted AtHIRD11 was retained in immobilized metal-affinity chromatography, and eluted by an imidazole gradient. The native AtHIRD11 prepared from Arabidopsis was partially phosphorylated, but further phosphorylated by casein kinase 2 in vitro. Metal-binding assays indicated that Zn²⁺ may be the best metal for AtHIRD11 binding. These results suggest that AtHIRD11 is a metal-binding dehydrin that shows a house-keeping expression in Arabidopsis.     

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.     

Suppression of oral tolerance by Lactococcus lactis in mice

Although oral ovabumin (OVA) administration suppressed the antibody (Ab) response in OVA-immunized mice, Lactococcus lactis increased OVA-specific IgG2a in these mice. L. lactis increased the casein-specific IgG level in NC/Nga mice fed on a casein diet. The percentage of CD4(+)CD25(+) cells was increased in DO11.10 mice orally given OVA, but this increase of CD4(+)CD25(+) cells were suppressed in L. lactis-fed DO11.10 mice.     

Prolonged, NK cell-mediated antitumor effects of suicide gene therapy combined with monocyte chemoattractant protein-1 against hepatocellular carcinoma

Tumor recurrence rates remain high after curative treatments for hepatocellular carcinoma (HCC). Immunomodulatory agents, including chemokines, are believed to enhance the antitumor effects of tumor cell apoptosis induced by suicide gene therapy. We therefore evaluated the immunomodulatory effects of a bicistronic recombinant adenovirus vector (rAd) expressing both HSV thymidine kinase and MCP-1 on HCC cells. Using an athymic nude mouse model (BALB/c-nu/nu), primary s.c. tumors (HuH7; human HCC cells) were completely eradicated by rAd followed by treatment with ganciclovir. The same animals were subsequently rechallenged with HCC cells, tumor development was monitored, and the recruitment or activation of NK cells was analyzed immunohistochemically or by measuring IFN-gamma mRNA expression. Tumor growth was markedly suppressed as compared with that in mice treated with a rAd expressing the HSV thymidine kinase gene alone (p < 0.001). Suppression of tumor growth was associated with the elevation of serum IL-12 and IL-18. During suppression, NK cells were recruited exclusively, and Th1 cytokine gene expression was enhanced in tumor tissues. The antitumor activity, however, was abolished either when the NK cells were inactivated with anti-asialo GM1 Ab or when anti-IL-12 and anti-IL-18 Abs were administered. These results indicate that suicide gene therapy, together with delivery of MCP-1, eradicates HCC cells and exerts prolonged NK cell-mediated antitumor effects in a model of HCC, suggesting a plausible strategy to prevent tumor recurrence.     

Eph receptors and ephrins

The Eph receptors are the largest known family of receptor tyrosine kinases. The Eph receptors and their membrane-attached ligands, ephrins, show diverse expression patterns during development. Recent studies have demonstrated that Eph receptors and ephrins play important roles in many developmental processes, including neuronal network formation, the patterning of the neural tube and the paraxial mesoderm, the guidance of cell migration, and vascular formation. In the nervous system, Eph receptors and ephrins have been shown to act as positional labels to establish topographic projections. They also play a key role in pathway finding by axons and neural crest cells. The crucial roles of Eph receptors and ephrins during development suggest involvement of these genes in congenital disorders affecting the nervous system and other tissues. It has also been suggested that Eph receptors and ephrins may be involved in carcinogenesis. It is therefore of clinical importance to further analyse the function of these molecules, as manipulation of their function may have therapeutic applications.     

The dynamic cycle of bacterial translation initiation factor IF3

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.     

Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1–like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.     

Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2^−/− mice adoptively transferred with CD4⁺CD45RB^high T cells; and IL-10^−/− mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b⁻CD11c^lowPDCA-1⁺ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4⁺CD45RB^high T cell-transferred RAG-2^−/− mice and IL-10^−/− mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4⁺ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation.     

Distribution and Respiratory Activity of Mycobacteria in Household Water System of Healthy Volunteers in Japan

The primary infectious source of nontuberculous mycobacteria (NTM), which are known as opportunistic pathogens, appears to be environmental exposure, and it is important to reduce the frequency of exposure from environmental sources for preventing NTM infections. In order to achieve this, the distribution and respiratory activity of NTM in the environments must be clarified. In this study, we determined the abundance of mycobacteria and respiratory active mycobacteria in the household water system of healthy volunteers using quantitative PCR and a fluorescent staining method, because household water has been considered as one of the possible infectious sources. We chose healthy volunteer households in order to lessen the effect of possible residential contamination from an infected patient. We evaluated whether each sampling site (bathroom drain, kitchen drain, bath heater pipe and showerhead) have the potential to be the sources of NTM infections. Our results indicated that drains in the bathroom and kitchen sink are the niche for Mycobacterium spp. and M. avium cells were only detected in the bathtub inlet. Both physicochemical and biologic selective pressures may affect the preferred habitat of Mycobacterium spp. Regional differences also appear to exist as demonstrated by the presence (US) or absence (Japan) of Mycobacterium spp. on showerheads. Understanding of the country specific human activities and water usage will help to elucidate the infectious source and route of nontuberculous mycobacterial disease.