Effects of elevated pressure on rate of photosynthesis during plant growth

The aim of this study is to investigate the effects of an artificially controlled environment, particularly elevated total pressure, on net photosynthesis and respiration during plant growth. Pressure directly affects not only cells and organelles in leaves but also the diffusion coefficients and degrees of solubility of CO₂ and O₂. In this study, the effects of elevated total pressure on the rates of net photosynthesis and respiration of a model plant, Arabidopsis thaliana, were investigated in a chamber that newly developed in this study to control the total pressure. The results clearly showed that the rate of respiration decreased linearly with increasing total pressure at a high humidity. The rate of respiration decreased linearly with increasing total pressure up to 0.2 MPa, and increased with increasing total pressure from 0.3 to 0.5 MPa at a low humidity. The rate of net photosynthesis decreased linearly with increasing total pressure under a constant partial pressure of CO₂ at 40 Pa. On the other hand, the rate of net photosynthesis was clearly increased by up to 1.6-fold with increasing total pressure and partial pressure of CO₂.     

Synthesis and biological evaluation of N-(2-fluorophenyl)-2β-deoxyfuconojirimycin acetamide as a potent inhibitor for α-l-fucosidases

In this study we revealed that the addition of an N-phenylacetamide substituent to the C-1 position of 1-deoxyfuconojirimycin (DFJ) can lead to highly potent inhibitors of α-l-fucosidases. A structure–activity relationship study showed that a fluoro group on the phenyl ring greatly increased its potency and selectivity. In contrast the addition of two or three fluoro groups decreased their inhibition potency. Consequently, N-(2-fluorophenyl)-2β-DFJ acetamide (18j) was found to display very potent and selective inhibition of bovine kidney, rat epididymis, and human lysosome α-l-fucosidases, with IC₅₀ value of 0.012, 0.044, and 0.0079 μM respectively. It is noteworthy that our designed N-phenyl-2β-DFJ acetamide derivative exhibited about 18-fold stronger effects on human lysosomal α-l-fucosidase than original DFJ and it occupied the active-site of this enzyme. We therefore expect that this compound may find applications in new therapeutic trials against genetic deficiency disorders.     

Involvement of membrane type 1-matrix metalloproteinase (MT1-MMP) in RAGE activation signaling pathways

An advanced glycation end products (AGE)/a receptor for AGE (RAGE) axis plays a key role in diabetic vascular complications. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown to function not only as a proteolytic enzyme but also as a signaling molecule. In this study, we investigated the role of MT1-MMP in the AGE/RAGE-triggered signaling pathways in cultured rabbit smooth muscle cells (SMCs) and the molecular interaction between RAGE and MT1-MMP in vitro and in vivo. In SMCs, AGE-activated Rac1 and p47(phox) within 1 min, NADPH oxidase activity and reactive oxygen species (ROS) generation within 5 min, and NF-κB phosphorylation within 15 min, thereby inducing redox-sensitive molecular expression. Silencing of RAGE by small-interfering RNA (siRNA) blocked the AGE-induced signaling pathways. AGE-induced geranylgeranyl transferase I (GGTase I) activity, Rac1·p47(phox) activation, NADPH oxidase activity, ROS generation, and molecular expression were also markedly attenuated by silencing of MT1-MMP. An inhibitor of GGTase I mimicked the effects of MT1-MMP-specific siRNA. Fluorescent immunohistochemistry revealed that MT1-MMP was partially co-localized with RAGE in SMCs, and RAGE was found to form a complex with MT1-MMP in both cultured SMCs and the aortae of diabetic rats by immunoprecipitation. Furthermore, MT1-MMP and RAGE formed a complex in the aortic atherosclerotic lesions of hyperlipidemic rabbits. We show that MT1-MMP plays a crucial role in RAGE-activated NADPH oxidase-dependent signaling pathways and forms a complex with RAGE in the vasculature, thus suggesting that MT1-MMP may be a novel therapeutic target for diabetic vascular complications.     

Receptor for Advanced Glycation End Products - Membrane Type1 Matrix Metalloproteinase Axis Regulates Tissue Factor Expression via RhoA and Rac1 Activation in High-Mobility Group Box-1 Stimulated Endothelial Cells

Background

Atherosclerosis is understood to be a blood vessel inflammation. High-mobility group box-1 (HMGB-1) plays a key role in the systemic inflammation. Tissue factor (TF) is known to lead to inflammation which promotes thrombus formation. Membrane type1 matrix metalloprotease (MT1-MMP) associates with advanced glycation endproducts (AGE) triggered-TF protein expression and phosphorylation of NF-κB. However, it is still unclear about the correlation of MT1-MMP and HMBG-1-mediated TF expression. In this study, we investigated the molecular mechanisms of TF expression in response to HMGB-1 stimulation and the involvement of MT1-MMP in endothelial cells.

Methods and Results

Pull-down assays and Western blotting revealed that HMGB-1 induced RhoA/Rac1 activation and NF-kB phosphorylation in cultured human aortic endothelial cells. HMGB-1 increased the activity of MT1-MMP, and inhibition of RAGE or MT1-MMP by siRNA suppressed HMGB-1-induced TF upregulation as well as HMGB-1-triggered RhoA/Rac1 activation and NF-kB phosphorylation.

Conclusions

The present study showed that RAGE/MT1-MMP axis modified HMBG-1-mediated TF expression through RhoA and Rac1 activation and NF-κB phosphorylation in endothelial cells. These results suggested that MT1-MMP was involved in vascular inflammation and might be a good target for treating atherosclerosis.     

Improvement of catechin productivity in suspension cultures of tea callus cells

In the suspension cultures of tea callus cells, C.sinensis cv. Yabukita, the effects of the culture conditions, such as culture period and light irradiation, on cell growth and catechin production were investigated. The production of flavonoids (catechins + proanthocyanidins) was promoted by inoculating the cells into the fresh medium at the culture period giving the maximum flavonoid content in the cells. The cultivation under light irradiation was repeated several times by inoculating the cells with the maximum flavonoid content. The flavonoid production was significantly increased without inhibiting the cell growth. We obtained the maximum flavonoid production, 1.5 g/dm(3) medium, and the maximum content, 150 mg/(g of dry cell weight (DCW)). The latter value was larger than that in the leaves of the tea plant.     

Remodeling of the left atrium in pacing-induced atrial cardiomyopathy

Rapid atrial pacing produces atrial systolic and diastolic failure characterized by absent atrial booster pump function, increased atrial chamber stiffness, enhanced atrial conduit function, and atrial enlargement. However, the processes underlying these abnormalities are poorly understood. Therefore, we examined left atrial myocardium from dogs with rapid pacing-induced atrial failure (400 bpm for 6 weeks) and from control dogs. Western blotting was used to determine the levels of proteins involved in calcium homeostasis (SERCA 2A, phospholamban, Na⁺-Ca²⁺ exchanger). Matrix metalloproteinase (MMP) activity was measured using gelatin and casein zymography, and levels of tissue inhibitor of metalloproteinase-4 (TIMP-4) and the TIMP-4 complexed with MMPs were measured with Western blot analysis. There were no differences in SERCA 2A or Na⁺-Ca²⁺ exchanger protein levels, but phospholamban level was significantly decreased in atrial samples from rapidly paced dogs (51.2 ± 7.8 vs. 77.0 ± 10.0, p < 0.01). The activity of MMP-9 was selectively and significantly increased by 50%, and the level of complexed TIMP-4 protein was significantly decreased by 50% in samples from dogs with atrial failure. Thus, rapid pacing-induced atrial failure is associated with differential changes in MMP activity, an unchanged number of calcium pumps, and compensatory changes in the level of phospholamban.     

Expression of interleukin-2 receptor (IL-2R) in feline peripheral blood lymphocytes

A monoclonal anti-interleukin-2 receptor (IL-2R) antibody has been identified as a putative antibody against the human IL-2R. In the present study, anti-Tac antibody CD-25 was used to determine cell expressing IL-2 receptor in feline peripheral blood lymphocytes by means of direct immunofluorescence tests and complement-mediated lymphocytotoxicity tests. With complement-mediated lymphocytotoxicity, approximately 18% of feline peripheral blood lymphocytes expressed the receptors. By the direct immunofluorescence test, we found approximately 22% of IL-2R positive cells in lymphocytes of feline peripheral-blood.     

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.     

Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18p11.32 by nonisotopic in situ hybridization

Summary The human thymidylate synthase (TS) gene was regionally assigned to chromosome band 18p11.32 by nonisotopic in situ hybridization using biotinylated cDNA (1.1kb insert) and genomic DNA (6.8kb insert) probes of the human gene. There have been two provisional assignments for the TS gene to 18pter-q12 and 18q21-qter. The present result confirmed the first of these and further localized the TS gene to the telomeric region of the short arm of chromosome 18. The TS gene appears to be a novel telomeric anchor point for the construction of both physical and genetic linkage maps of human chromosome 18.     

Unusual aspects of human thymidylate synthase in mouse cells introduced by DNA-mediated gene transfer

Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme. In two transformants examined, however, neither was the dTTP pool elevated nor the dCTP pool decreased. DNA synthesis in permeabilized cells of a transformant was more efficient than that in the wild type with dATP, dGTP, dCTP, and dUMP as substrates, but this was not so when dUMP was replaced by dTTP. Unlike the mouse enzyme, the human enzyme in the transformants did not co-sediment with DNA polymerase alpha and thymidine kinase in a sucrose gradient, suggesting that the human enzyme is not incorporated into a multienzyme complex for DNA replication. The high levels of the human enzyme in the transformants were suppressed to various degrees by fusion with a wild type mouse line. No active hybrid dimer enzyme was found between the human and mouse enzymes, which each consist of two identical subunits. Thus, the human enzyme in the transformants seems to behave differently from the mouse enzyme and its overproduction seems to be necessary for supporting the normal growth of the transformants.