[Effect of different mechanical loading on the expression of Notch signaling pathways in growing rabbits' condylar cartilage].

Objective: To investigate the effect of different mechanical loading on the expression of Notch-1, Notch-3, Jagged-1 and Delta-like-1 in growing rabbits' condylar cartilage. Methods: Sixty-four ten-days-old rabbits were randomly divided into experimental and control groups. The rabbits in the experimental groups were fed on a powder diet, while the control groups were fed on a solid diet. The animals were sacrificed after 2, 4, 6 and 8 weeks. Notch-1, Notch-3, Jagged-1 and Delta-like-1 gene and protein expressions were examined by HE, immunohistochemistry, Western blotting and quantitative real-time PCR. Results: At 2, 4, 6, 8 weeks, the anterior part of condylar cartilage in solid diet groups ([318.1±4.3], [342.4±2.6], [364.2±3.2], [380.7±6.0] mm, respectively) were thicker than those in powder diet groups ([275.4±2.6], [301.1±2.0], [322.3±3.3], [366.5±8.4] mm, respectively) (P<0.05). There was no significant difference between the two groups in the middle part (P>0.05). From 2 to 6 weeks, the posterior part of cartilage in solid diet groups ([444.1±1.5], [451.1±0.3], [476.4±5.7] mm, respectively) was thinner than those in power diet groups ([470.4±2.7], [494.3±2.9], [512.3±5.7] mm, respectively) (P<0.05). At 6 weeks, there were more mRNA expressions of Notch-3, Jagged-1 and Delta-like-1 in solid diet group than in power diet group (P<0.05). The Notch-1 protein could be found in all layers of condylar cartilage, especially in the surface of hypertrophic zone. The expressions of Notch-1, Notch-3, and Delta-like-1 protein were increased from 2 to 6 weeks, and decreased at 8 weeks. In solid diet groups, the protein expressions of Notch-1 (at 2, 4, 6, 8 weeks), Notch-3 (at 2, 4, 6 weeks), Jagged-1 (at 2, 4, 6 weeks) and Delta-like-1 (at 4, 6, 8 weeks) were greater than in power diet groups (P<0.05). Conclusions: Low masticatory loading may delay or inhibit the development of condylar cartilage and its growing factors such as Notch-1, Notch-3, Jagged-1 and Delta-like-1. Appropriate masticatory loading plays an important role in normal development of the condyle.

Azaphilones from an Acid Mine Extremophile Strain of a Pleurostomophora sp.

An extremophilic fungus identified as a Pleurostomophora sp. was isolated from the Berkeley Pit, an acid mine waste lake. When grown in liquid culture, the fungus produced berkchaetoazaphilones A-C (1, 2, and 5), the red pigment berkchaetorubramine (6), and the known compound 4-(hydroxymethyl)quinoline. These compounds were evaluated as inhibitors of matrix metalloproteinase-3, caspase-1, and proinflammatory cytokine production in induced THP-1 cells. Berkchaetoazaphilone B (2) inhibited IL-1ß, TNFα, and IL-6 production in the induced inflammasome assay and was cytotoxic toward human retinoblastoma cell line Y79 (IC50 = 1.1 µM), leukemia cell lines CCRF-CEM and SR, and the melanoma cell line LOX IMVI (IC50 = 10 µM).

Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA.

The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.

The binding affinity of Ff gene 5 protein depends on the nearest-neighbor composition of the ssDNA substrate.

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.

Suppression of nitric oxide-induced apoptosis by N-acetyl-L-cysteine through modulation of glutathione, bcl-2, and bax protein levels.

It has been demonstrated that nitric oxide (NO) can promote apoptosis in human cancer cells. To test the protective effects of antioxidants (N-acetyl-L-cysteine (LNAC) and free-radical spin traps (5,5-dimethyl-1-pyrroline N-oxide and 2,2,6,6,-tetramethyl-1-piperidinyloxy) against NO-induced apoptosis, a human colon cancer cell line (COLO 205) was treated with NO, and its survival rate was evaluated both with and without antioxidant therapy. LNAC arrested the development of progression of apoptosis in COLO 205 cells in a dose-dependent manner, promoted long-term survival, and prevented the internucleosomal DNA cleavage induced by NO. The intracellular level of glutathione (GSH) was found to be elevated in cells after exposure to LNAC. The bax protein levels were elevated by NO treatment, and this effect was blocked by LNAC. On the other hand, the bcl-2 oncoprotein level in the LNAC-pretreated cells was significantly elevated in a time-dependent manner compared to cells that received NO pretreatment. In summary, our results suggest that the protective effect of LNAC may be linked to its inducement of increases in cellular GSH and bcl-2 protein levels and to its suppression of cellular bax protein in treated cells.