Polarity of response to transforming growth factor-beta1 in proximal tubular epithelial cells is regulated by beta-catenin

Transforming growth factor-beta1 (TGF-beta1)-mediated loss of proximal tubular epithelial cell-cell interaction is regulated in a polarized fashion. The aim of this study was to further explore the polarity of the TGF-beta1 response and to determine the significance of R-Smad-beta-catenin association previously demonstrated to accompany adherens junction disassembly. Smad3 signaling response to TGF-beta1 was assessed by activity of the Smad3-responsive reporter gene construct (SBE)(4)-Lux and by immunoblotting for phospho-Smad proteins. Similar results were obtained with both methods. Apical application of TGF-beta1 led to increased Smad3 signaling compared with basolateral stimulation. Association of Smad proteins with beta-catenin was greater following basolateral TGFbeta-1 stimulation, as was the expression of cytoplasmic Triton-soluble beta-catenin. Inhibition of beta-catenin expression by small interfering RNA augmented Smad3 signaling. Lithium chloride, a GSK-3 inhibitor, increased expression of beta-catenin and attenuated TGF-beta1-dependent Smad3 signaling. Lithium chloride did not influence degradation of Smad3 but resulted in decreased nuclear translocation. Smad2 activation as assessed by Western blot analysis and activity of the Smad2-responsive reporter constructs ARE/MF1 was also greater following apical as compared with basolateral TGFbeta-1 stimulation, suggesting that this is a generally applicable mechanism for the regulation of TGF-beta1-dependent R-Smads. Caco-2 cells are a colonic carcinoma cell line, with known resistance to the anti-proliferative effects of TGF-beta1 and increased expression of beta-catenin. We used this cell line to address the general applicability of our observations. Inhibition of beta-catenin in this cell line by small interfering RNA resulted in increased TGF-beta1-dependent Smad3 phosphorylation and restoration of TGF-beta1 anti-proliferative effects.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

Trivalent recognition unit of innate immunity system: crystal structure of trimeric human M-ficolin fibrinogen-like domain

Ficolins are a kind of pathogen-recognition molecule in the innate immune systems. To investigate the discrimination mechanism between self and non-self by ficolins, we determined the crystal structure of the human M-ficolin fibrinogen-like domain (FD1), which is the ligand-binding domain, at 1.9A resolution. Although the FD1 monomer shares a common fold with the fibrinogen gamma fragment and tachylectin-5A, the Asp-282-Cys-283 peptide bond, which is the predicted ligand-binding site on the C-terminal P domain, is a normal trans bond, unlike the cases of the other two proteins. The trimeric formation of FD1 results in the separation of the three P domains, and the spatial arrangement of the three predicted ligand-binding sites on the trimer is very similar to that of the trimeric collectin, indicating that such an arrangement is generally required for pathogen-recognition. The ligand binding study of FD1 in solution indicated that the recombinant protein binds to N-acetyl-d-glucosamine and the peptide Gly-Pro-Arg-Pro and suggested that the ligand-binding region exhibits a conformational equilibrium involving cis-trans isomerization of the Asp-282-Cys-283 peptide bond. The crystal structure and the ligand binding study of FD1 provide an insight of the self- and non-self discrimination mechanism by ficolins.     

Temporal changes in the growth, saponin content and antioxidant defense in the adventitious roots of Panax ginseng subjected to nitric oxide elicitation

Nitric oxide (NO) is a diffusible, gaseous signaling molecule. In plants, NO influences growth and development, and it can also affect plant responses to various stresses. Because NO induces root differentiation and interacts with reactive oxygen species, we examined the temporal effect of NO elicitation on root growth, saponin accumulation and antioxidant defense responses in the adventitious roots of mountain ginseng (Panax ginseng). The observations revealed that NO is involved in root growth and saponin production. Elicitation with sodium nitroprusside (SNP) activated O₂ ⁻-generating NADPH oxidase (NOX) activity, which most probably subsequently enhanced growth of adventitious roots of mountain ginseng. A severe inhibition of NOX activity and decline in dry weight of SNP elicited adventitious roots in the presence of NOX inhibitor (diphenyl iodonium, DPI), which further supports involvement of NOX in root growth. Enhanced activities of antioxidant enzymes by SNP appear to be responsible for low H₂O₂, less lipid peroxidation, and modulation of ascorbate and non-protein thiol statuses in the adventitious roots of mountain ginseng. Dry mass, saponin content and NOX activity was related with NO content present in adventitious roots of mountain ginseng.     

Potential resistance of tricin in rice against brown planthopper Nilaparvata lugens (Stål)

The aerial parts of resistant rice plant (IR36) was immersed in 85% ethanol at room temperature, and ethanol-soluble extracts were prepared. Different solvents including petroleum ether, ethyl acetate, absolute ethanol, and water were then used to separate the ethanol-soluble extracts and different solvent fractions were obtained. These fractions were tested for their effects on the mortality rate of the brown planthopper nymphae (BPH). The results indicated that EtOAc fractions had significant effects on the mortality of brown planthopper nymphae after the third day of treatment. EtOAc fractions of resistant rice plants (IR36) were separated using column chromatography over silica gel and 5,7,4′ -trihydroxy-3′,5′-dimethoxyflavone (tricin) was isolated. After the BPH were allowed to feed on diets containing tricin for 15 days, it was found that the weight of honeydew of the nymphae showed a decrease as tricin concentrations in the diets increased. The mortality rate of the nymphae increased with increasing tricin concentration. The corrected mortality rate was 58.21% for the 3rd instar nymphae and 31.75% for the 4th instar nymphae, respectively, at 500 μg/ml concentration. In addition, tricin (500 μg/ml) significantly inhibited oviposition behavior and feeding of the brown planthopper females in choice tests when spread on susceptible rice plant (TN1).     

Effective tempo of the step test for dynamic balance ability in the elderly

This study aimed to examine the reliability and sex- and age-related differences of step tests with stipulated tempos as well as to clarify useful test parameters and tempos. One hundred forty elderly people and fifty young adults conducted tapping and stepping tests, matching the tempo provided by a metronome. Both tests involve movements where the subject touches a sheet with both the right and left hands or right and left legs at a designated spot. Evaluation parameters were the time difference between the beep sound and the time at which the sheet was touched in both tests as well as two-leg support and one-leg support times in the step test. The trial-to-trial reliability of the parameters in both tests was high. The time differences of both 40 bpm tests in the elderly were larger in males than in females. In the step test, the time difference and two-leg support times of the elderly were larger, in the order of 40, 60, and 120 bpm, and the one-leg support time was less in 40 bpm than 60 bpm or 120 bpm. The one-leg support time of the young subjects was larger, in the order of 40, 60, and 120 bpm. A significant age-related difference was found in the 40 bpm and 60 bpm test, and the time difference and two-leg support time were larger in the elderly while the one-leg support time was larger in the young subjects. The time difference at 40 bpm in the elderly was larger in the step test than in the tap test. There was no significant difference between both tests in the young subjects. In conclusion, the step test with the slow tempo, because it requires a long one-leg support phase, is effective for evaluating dynamic balance in the elderly. The time difference and two-leg and one-leg support times are effective evaluation parameters of the step test.     

Single feature polymorphism discovery in rice

The discovery of nucleotide diversity captured as single feature polymorphism (SFP) by using the expression array is a high-throughput and effective method in detecting genome-wide polymorphism. The efficacy of such method was tested in rice, and the results presented in the paper indicate high sensitivity in predicting SFP. The sensitivity of polymorphism detection was further demonstrated by the fact that no biasness was observed in detecting SFP with either single or multiple nucleotide polymorphisms. The high density SFP data that can be generated quite effectively by the current method has promise for high resolution genetic mapping studies, as physical location of features are well-defined on rice genome.