Structural and biochemical analyses of hemimethylated DNA binding by the SeqA protein

The Escherichia coli SeqA protein recognizes the 11 hemimethylated G-^mA-T-C sites in the oriC region of the chromosome, and prevents replication over-initiation within one cell cycle. The crystal structure of the SeqA C-terminal domain with hemimethylated DNA revealed the N⁶-methyladenine recognition mechanism; however, the mechanism of discrimination between the hemimethylated and fully methylated states has remained elusive. In the present study, we performed mutational analyses of hemimethylated G-^mA-T-C sequences with the minimal DNA-binding domain of SeqA (SeqA_71–181), and found that SeqA_71–181 specifically binds to hemimethylated DNA containing a sequence with a mismatched ^mA:G base pair [G-^mA(:G)-T-C] as efficiently as the normal hemimethylated G-^mA(:T)-T-C sequence. We determined the crystal structures of SeqA_71–181 complexed with the mismatched and normal hemimethylated DNAs at 2.5 and 3.0 Å resolutions, respectively, and found that the mismatched ^mA:G base pair and the normal ^mA:T base pair are recognized by SeqA in a similar manner. Furthermore, in both crystal structures, an electron density is present near the unmethylated adenine, which is only methylated in the fully methylated state. This electron density, which may be due to a water molecule or a metal ion, can exist in the hemimethylated state, but not in the fully methylated state, because of steric clash with the additional methyl group.     

Imaging analysis of subcellular correlation of androgen receptor and estrogen receptor alpha in single living cells using green fluorescent protein color variants

Androgen and estrogen act not only in a sex-specific manner but also interactively and synergistically. In the present study, to examine the possible interaction between androgen receptor (AR) and estrogen receptor-alpha (ERalpha), we investigated the subcellular dynamics of AR and ERalpha fused with green fluorescent protein color variants in single living cells using time-lapse microscopy and the technique of fluorescence recovery after photobleaching. AR and ERalpha showed punctate colocalization in the nucleus with estrogen, but not androgen. N-terminal AR deletion mutant did not form a nuclear punctate pattern with either androgen or estrogen. In the presence of AR, but not ERalpha, N-terminal AR deletion mutant formed a punctate nuclear pattern with androgen. AR had different mobility depending on the ligand and the presence of ERalpha. On the other hand, AR had little effect on the stability of ERalpha. ERalpha mutant that does not bind coactivators did not alter the mobility of AR. Taken together, using an imaging technique, we clarified that possible homo/hetero dimerization between AR and ERalpha could be attributed to androgen-estrogen interaction in living cells.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole induces apoptosis and necrosis with activation of different caspases in rat splenocytes

A dietary carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) at 20 microM activates caspase-3-like proteases as an apoptotic marker in rat splenocytes. The present study demonstrated 100 microM Trp-P-1 induced necrosis with activation of caspase-3-like proteases. The activation in necrosis and apoptosis resulted from the activation of caspase-9 and caspase-8, respectively. Thus, Trp-P-1 induces apoptosis and necrosis with the activation of different caspases.     

Enhanced calcium absorption in the small intestine by a phytate-removed deamidated soybean globulin preparation

Soybean globulins were deamidated after removing phytate using ion-exchange resins, and then hydrolyzed by digestive enzymes. The phytate-removed deamidated soybean globulins (PrDS) retained high calcium-binding ability even after the hydrolysis by digestive enzymes. PrDS and its hydrolysates enhanced calcium absorption from the small intestine when injected into the small intestine together with a calcium solution.     

Effect of phytate-removal and deamidation of soybean proteins on calcium absorption in the in situ rats

Soybean proteins were deamidated by cation-exchange resins after phytate, the inhibitor for calcium absorption from the small intestine, was removed in order to provide the enhancement function of calcium absorption to soybean proteins. About 92% of the phosphorus was removed from the soybean proteins by anion-exchange-resin treatment, indicating that most of the phytate was removed. About 70% of the acid amide was deamidated by cation-exchange-resin treatment, and phytate-removed and deamidated soybean proteins (PrDS) having high calcium binding properties were obtained. PrDS were hydrolyzed by digestive enzymes and their calcium-binding properties and the enhancement function of the calcium absorption from the small intestine of rats were examined. As a result, PrDS retained their high calcium binding properties even after hydrolysis by digestive enzymes. In situ experiments showed that PrDS and their hydrolysates enhanced the calcium absorption from the intestine.     

DNA chip analysis of comprehensive food function: inhibition of angiogenesis and telomerase activity with unsaturated vitamin E, tocotrienol

Inhibition of angiogenesis and telomerase activity with vitamin E compounds, especially for tocotrienol (T3), has been investigated. Nutrigenomic tools have been used for elucidating the bioactive mechanisms of T3. In the cell culture experiments, T3 reduced the vascular endothelial growth factor (VEGF)-stimulated tube formation by human umbilical vein endothelial cells (HUVEC). Among T3 isomers, delta-T3 appeared the highest activity. The T3 inhibited the new blood vessels formation on the growing chick embryo chorioallantoic membrane (CAM assay for an in vivo model of angiogenesis). In contrast, tocopherol did not. The findings suggested that the T3 has potential use for reducing angiogenic disorder. DNA chip analysis revealed that T3 specifically down-regulates the expression of VEGF receptor (VEGFR) in endothelial cells. It is well-known that VEGF regulates angiogenesis by binding to VEGFR. Therefore, T3 could block the intracellular signaling of VEGF via down-regulation of VEGFR, which resulted in the inhibition of angiogenesis. On the other hand, DNA chip analysis also revealed that T3 down-regulates the expression of protein kinase C (PKC) in the cultured HUVEC. Since PKC is involved with the control of telomerase activity, T3 has potential to act as anti-telomerase inhibitor via PKC inhibition. In this manner, DNA chip technology provides efficient access to genetic information regarding food function and its mechanism.     

Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity

c-Jun NH₂-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle. electroporation; gene delivery; muscle contraction; exercise     

ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase

The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.     

Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.