Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.     

Psicose inhibits lettuce root growth via a hexokinase-independent pathway

Fructose analog, psicose, and glucose analog, mannose, inhibited root growth of lettuce seedlings. Psicose is phosphorylated by hexokinase and fructokinase (EC 2.7.1.4) to psicose-6-phosphate with no known capacity for further metabolism. Mannose is phosphorylated by hexokinase (EC 2.7.1.1) to mannose-6-phosphate which is further metabolized very slowly. Hexokinase is known to have a sugar-sensing function and possibly triggers a signal cascade resulting in changes of several gene expressions. It was determined, compared with the behaviour of mannose, whether psicose inhibits the root growth through this system. The addition of phosphate into the growth medium of lettuce seedlings did not affect the inhibition by psicose and mannose, and both sugars did not reduce adenosine triphosphate (ATP) level in the roots, suggesting that the inhibition is not due to phosphate starvation and ATP depletion. The inhibiting effects of psicose and mannose were overcome by adding sucrose into the medium, which suggests that the inhibition is not caused by accumulation of psicose-6-phosphate or mannose-6-phosphate in the seedlings. Mannoheptulose, a specific competitive inhibitor of hexokinase, defeated the mannose-induced inhibiting but was not able to relieve the psicose-induced inhibition. Thus, the phosphorylation of mannose by hexokinase may trigger a signal cascade resulting in the growth inhibition of lettuce roots, which is consistent with the hypothesis established in Arabidopsis . However, psicose cannot inhibit the growth of lettuce roots via a hexokinase-mediated pathway, and the phosphorylation of psicose by fructokinase might trigger a hexokinase-independent signal cascade resulting in the growth inhibition.     

Effect of the structure of dietary epoxylignan on its cytotoxic activity: relationship between the structure and the activity of 7,7′-epoxylignan and the introduction of apoptosis by caspase 3/7

We compared the cytotoxic activities of dietary epoxylignans and their stereoisomers and found (?)-verrucosin, which is (7S,7′R,8R,8′R)-7,7′-epoxylignan, to be the most cytotoxic epoxylignan against HeLa cells (IC₅₀ = 6.6 μM). On the other hand, the activity was about a factor of 10 less against HL-60. In this research on the relationship between the structure and cytotoxic activity of (?)-verrucosin 13, the 7-(4-methoxyphenyl)-7′-(3,4-dimethoxyphenyl) derivative 60, for which the activity (IC₅₀ = 2.4 μM) is three times greater than (?)-verrucosin 13, was discovered. The induction of apoptosis by caspase 3/7 was observed upon treatment with the (?)-verrucosin derivative.     

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.     

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N₂O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO₂^?) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.     

Worm wounding increases levels of pollen-related food allergens in soybean (Glycine max)

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30 K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans.     

Characterization of a novel hydroxynitrile lyase from Nandina domestica Thunb

The leaves of Nandina domestica Thunb. exhibited high hydroxynitrile lyase (HNL) activity in (R)-mandelonitrile synthesis. The specific activity of young leaves was significantly higher than that of mature leaves. We isolated two HNLs with molecular mass of 24.9 kDa (NdHNL-S) and 28.0 kDa (NdHNL-L) from the young leaves. Both NdHNLs were composed of two identical subunits, without FAD and carbohydrates. We purified NdHNL-L and revealed its enzymatic properties. The whole deduced amino acid sequence of NdHNL-L was not homologous to any other HNLs, and the specific activity for mandelonitrile synthesis by NdHNL-L was higher than that by other plant HNLs. The enzyme catalyzed enantioselective synthesis of (R)-cyanohydrins, exhibited high activity at pH 4.0, and high stability in the pH range of 3.5–8.0 and below 55°C. Thus, NdHNL-L is a novel HNL with novel amino acid sequence and has a potential for the efficient production of (R)-cyanohydrins.     

CpdA is involved in amino acid metabolism in Shewanella oneidensis MR-1

Cyclic 3′,5′-adenosine monophosphate (cAMP) phosphodiesterase (CPD) is an enzyme that catalyzes the hydrolysis of cAMP, a signaling molecule affecting diverse cellular and metabolic processes in bacteria. Some CPDs are also known to function in cAMP-independent manners, while their physiological roles remain largely unknown. Here, we investigated physiological roles of CPD in Shewanella oneidensis MR-1, a model environmental bacterium, and report that CPD is involved in amino-acid metabolism. We found that a CPD-deficient mutant of MR-1 (ΔcpdA) showed decreased expression of genes for the synthesis of methionine, S-adenosylmethionine, and histidine and required these three compounds to grow in minimal media. Interestingly, deletion of adenylate cyclases in ΔcpdA did not restore the ability to grow in minimal media, indicating that the amino acid requirements were not due to the accumulation of cAMP. These results suggest that CPD is involved in the regulation of amino acid metabolism in MR-1 in a cAMP-independent manner.