Virus-induced gene silencing of <Emphasis Type="Italic">14-3-3</Emphasis> genes abrogates dark repression of nitrate reductase activity in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

In order to study the effect of repression of 14-3-3 genes on actual activity of the nitrate reductase (NR) in Nicotiana benthamiana leaves, Nb14-3-3a gene was silenced by virus-induced gene silencing (VIGS) method using potato virus X (PVX). Expression of Nb14-3-3a as well as Nb14-3-3b genes was altogether repressed in the leaves of PVX-14-3a-infected plants. Furthermore, two-dimensional gel electrophoresis and immunoblot analysis with anti-14-3-3 antiserum suggested that the expressions of Nb14-3-3a and Nb14-3-3b proteins are accordingly repressed in PVX-14-3a-infected plants. It is well known that binding of 14-3-3 proteins to phosphorylated NR leads to substantial decrease in NR activity of leaves under darkness. Therefore, we studied the changes in NR activity in response to light/dark transitions in the leaves of PVX-14-3a-infected plants. NR activation state was kept at a high level under darkness in PVX-14-3a-infected plants, but not in PVX-green fluorescent protein (GFP)-infected and control plants. This result suggests that Nb14-3-3a and/or Nb14-3-3b proteins are indeed involved in the inactivation of NR activity under darkness in N. benthamiana.     

In vitro evaluation of tipburn resistance in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis>. L)

Lettuce tipburn is an irreversible physiological disorder caused by calcium deficiency that decreases the crop value. Breeding a tipburn-resistant cultivar is the only causal therapy in many cases. In this study, we investigated an efficient method to evaluate lettuce resistance to tipburn in vitro. Seedlings of 19 lettuce cultivars representing three head types were cultured on agar medium containing EGTA, which chelates Ca²⁺. The percentage of tipburned leaves decreased proportionally with EGTA concentration. Susceptible cultivars were distinguished at 0.01 mM EGTA, whereas resistant cultivars were classified at 1.0 mM EGTA. Based on mean values of tipburn measurements, tipburn susceptibility was highest for ‘Leaf Lettuce’, followed by ‘Butterhead Lettuce’, and then ‘Crisphead Lettuce’. Two cultivars were selected for further tests using hydroponic and pot culture. The rank order of susceptibility to tipburn in these experiments was consistent with that of the in vitro assay. The in vitro evaluation of lettuce susceptibility to calcium deficiency is useful for initial screening of lettuce cultivars against tipburn incidence. Resistant cultivars identified in this study are practical candidates for cultivation in controlled environments, such as a plant factory, while sensitive cultivars are also useful as indicator plants to monitor environmental conditions.     

Novel properties of statins: suppression of the systemic and bone marrow responses induced by exposure to ambient particulate matter (PM10) air pollution

Exposure to ambient particulate matter (PM(10)) elicits systemic inflammatory responses that include the stimulation of bone marrow and progression of atherosclerosis. The present study was designed to assess the effect of repeated exposure of PM(10) on the turnover and release of polymorphonuclear leukocytes (PMNs) from the bone marrow into the circulation and the effect of lovastatin on the PM(10)-induced bone marrow stimulation. Rabbits exposed to PM(10) three times a week for 3 wk, were given a bolus of 5'-bromo-2'-deoxyuridine to label dividing cells in the marrow to calculate the transit time of PMNs in the mitotic or postmitotic pool. PM(10) exposure accelerated the turnover of PMNs by shortening their transit time through the marrow (64.8 ± 1.9 h vs. 34.3 ± 7.4 h, P < 0.001, control vs. PM(10)). This was predominantly due to a rapid transit of PMNs through the postmitotic pool (47.9 ± 0.7 h vs. 21.3 ± 4.3 h, P < 0.001, control vs. PM(10)) but not through the mitotic pool. Lovastatin delayed the transit time of postmitotic PMNs (38.2 ± 0.5 h, P < 0.001 vs. PM(10)) and shifted the postmitotic PMN release peak from 30 h to 48 h. PM(10) exposure induced the prolonged retention of newly released PMNs in the lung, which was reduced by lovastatin (P < 0.01). PM(10) exposure increased plasma interleukin-6 levels with significant reduction by lovastatin (P < 0.01). We conclude that lovastatin downregulates the PM(10)-induced overactive bone marrow by attenuating PM(10)-induced systemic inflammatory responses.     

Involvement of dynamin-2 in formation of discoid vesicles in urinary bladder umbrella cells

Umbrella cells (UCs) of the epithelium of the urinary bladder have the capacity to control bladder volume by regulating exocytosis/endocytosis of their intracellular discoid vesicles (DVs). Dynamin (Dyn) is a GTPase that promotes endocytic processes through scission of cell membranes. We have examined whether Dyn2, the most abundant Dyn form, is expressed in UCs and contributes to their endocytic actions. A specific antibody against Dyn2 was used to localize Dyn2 in human and rodent UCs by immunohistochemistry. To clarify the functional roles of Dyn2, mouse bladders were treated with a Dyn-GTPase inhibitor, dynasore, and its effects on their UC structure were assessed. Since uropathogenic Escherichia coli can be encased into UCs during infection, we used immunohistochemistry to determine whether bacteria-encasing compartments in the infected UCs were also enriched with Dyn2. Light microscopy showed that Dyn2 was abundantly expressed in UCs, especially near the apical cytoplasmic regions. By immunoelectron microscopy, Dyn2 was found on and around DV membranes in UCs. Ultrastructural analysis with a quick-freezing and deep-etching method confirmed these findings and revealed the existence of distinct Dyn2-bound microfilaments in close association with DV membranes. Dynasore treatment of bladders markedly reduced the number of DVs in UCs. In infected UCs, E. coli was encased in compartments enriched in Dyn2. Therefore, Dyn2 is highly enriched in UCs and mostly associated with membranes of DVs and microfilaments in the UCs. Pretreatment of bladders with dynasore inhibits E. coli invasion of UCs. Dyn2 thus contributes to the structural integrity of DVs and to the endocytic activity of UCs.     

Trans-splicing as a novel method to rapidly produce antibody fusion proteins

To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V_H-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to Sμ as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since Sμ sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).     

Regulation by sphingolipids of the fate of FRTL-5 cells

Sphingolipids, including ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1-P) have recently emerged as signal-transducing molecules. Functionally, a distinguishing characteristic of these lipids is their apparent participation in pro- or anti-proliferative cell regulation pathways. In this study, we examined the involvement of sphingolipids in the fate of FRTL-5 thyroid follicular cells. We first examined the effects of sphingolipids on FRTL-5 cell viability. Sph and Cer induced apoptosis, as revealed by fluorescence microscopy of TUNEL-positive fragmented nuclei and 180-300 bp DNA fragmentation on agarose gel electrophoresis while Sph-1-P was confirmed to prevent FRTL-5 cell apoptosis induced by deprivation of serum and TSH, possibly via cell surface receptors. We then analysed the metabolism of radiolabelled Sph and C(6)-Cer (a synthetic cell-permeable Cer) in FRTL-5 cells by thin layer chromatography, followed by autoradiography. Sph was mainly metabolized to Cer, and then to sphingomyelin, while Sph conversion into Sph-1-P was hardly detected. These changes were not affected by stimulation of the cells with TSH. Our results indicate the involvement of sphingolipid mediators in the fate of FRTL-5 thyroid cells.     

A high‐fat diet exacerbates the Alzheimer's disease pathology in the hippocampus of the App NL−F/NL−F knock‐in mouse model

Insulin resistance and diabetes mellitus are major risk factors for Alzheimer''s disease (AD), and studies with transgenic mouse models of AD have provided supportive evidence with some controversies. To overcome potential artifacts derived from transgenes, we used a knock‐in mouse model, App^{NL−F/NL−F} , which accumulates Aβ plaques from 6 months of age and shows mild cognitive impairment at 18 months of age, without the overproduction of APP. In the present study, 6‐month‐old male App^{NL−F/NL−F} and wild‐type mice were fed a regular or high‐fat diet (HFD) for 12 months. HFD treatment caused obesity and impaired glucose tolerance (i.e., T2DM conditions) in both wild‐type and App^{NL−F/NL−F} mice, but only the latter animals exhibited an impaired cognitive function accompanied by marked increases in both Aβ deposition and microgliosis as well as insulin resistance in the hippocampus. Furthermore, HFD‐fed App^{NL−F/NL−F} mice exhibited a significant decrease in volume of the granule cell layer in the dentate gyrus and an increased accumulation of 8‐oxoguanine, an oxidized guanine base, in the nuclei of granule cells. Gene expression profiling by microarrays revealed that the populations of the cell types in hippocampus were not significantly different between the two mouse lines, regardless of the diet. In addition, HFD treatment decreased the expression of the Aβ binding protein transthyretin (TTR) in App^{NL−F/NL−F} mice, suggesting that the depletion of TTR underlies the increased Aβ deposition in the hippocampus of HFD‐fed App^{NL−F/NL−F} mice.     

Novel pyridobenzimidazole derivatives exhibiting antifungal activity by the inhibition of β-1,6-glucan synthesis

Based on the HTS hit compound 1a, an inhibitor of β-1,6-glucan synthesis, we synthesized novel pyridobenzimidazole derivatives and evaluated their antifungal activity. Among the compounds synthesized, we identified the potent compound 15e, which exhibits excellent activity superior to fluconazole against both Candida glabrata and Candida krusei. From the SAR study, we revealed essential moieties for antifungal activity.