Structures of four-way junctions in gene <i>17</i> of T3 and T7.

<p>(A) T3 nt 33324–33353 (B) T3 nt 33317–33347 (C) T7 nt 35109–35139 (D) T7 nt 35082–35117. One strand is highlighted in grey; the other is not. Arrows indicate Endo I cutting sites.</p

Efficiency of plating of phages on <i>E. coli</i> female and male strains determined at 37°C.

<p>Efficiency of plating of phages on <i>E. coli</i> female and male strains determined at 37°C.</p

Alignment of T3 and T7 sequences near the crossover regions seen in the T3/7 phage.

<p>(A) Within gene <i>17</i>. (B) Within gene <i>18.5</i>. Sequences were aligned by ClustalW. The parent phages of the two sides of the crossover region (shown by arrows) are indicated on top of the alignment.</p

Mapping of the T3/7 and T7M DNA.

<p>The genomic DNA was digested by restriction endonucleases, and the fragments were observed by 1% (T3/7) or 0.8% (T7M) agarose gel electrophoresis. M: DNA marker. (A) Digestions of T3/7 DNA. Lane 1, <i>Mbo</i>I; lane 2, <i>Hpa</i>I; lane 3, <i>Nde</i>I; lane 4, <i>Stu</i>I; lane 5, <i>Avr</i>II. The markers are the same for the three slices of gels. (B) Digestion of T7M DNA. Lane 1, <i>Hpa</i>I. (C) Restriction sites in the T3/7 genome. Restriction sites are represented by vertical bars above the DNA. A: The T3 genome (black) and the region replaced by T7 DNA (in grey) in T3/7. The region of recombination is deduced from restriction mapping and sequencing. The two <i>Mbo</i>I sites in T7 that replace the single <i>Mbo</i>I site at T3 nt 34033 are shown by vertical bars. B to E show restriction sites in T3 genome (black); a dot above the bar indicates that the cutting site is present in T3 but not in T3/7. B: <i>Hpa</i>I sites, C: <i>Mbo</i>I sites, D: <i>Nde</i>I sites, E: <i>Avr</i>II cutting positions are indicated by bars, and the single <i>Stu</i>I site is shown by an arrow.</p

The mechanism of endonucleolytic cleavages at nonequivalent sites and strand annealing (CNSA).

<p>Colors and arrows are the same as those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030954#pone-0030954-g005" target="_blank">Figure 5</a>. The steps are: 1, production of DSBs by cutting at nonequivalent sites of T3 and T7 DNA; 2, 5′ resections of DSBs of T3 and T7; 3, annealing of the T3 DSBs with the T7 DSB; 4, removal of the nonhomologous nucleotides, filling the gap, and ligation.</p

Structures of four-way junctions in gene <i>18.5</i> of T3 and T7.

<p>(A) T3 nt 35315–35345 or equivalently T7 nt 37094–37124 (B) T7 nt 37163–37188. One of the strands is highlighted in grey. Endo I cutting sites are shown by arrows.</p

Adsorption efficiency (%) of phages on <i>E. coli</i> female and male strains.

<p>Adsorption efficiency (%) of phages on <i>E. coli</i> female and male strains.</p

Upregulation of Bone Morphogenetic Protein-2 Synthesis and Consequent Collagen II Expression in Leptin-stimulated Human Chondrocytes

<div><p>Bone morphogenetic proteins (BMPs) play positive roles in cartilage development, but they can barely be detected in healthy articular cartilage. However, recent evidence has indicated that BMPs could be detected in osteoarthritic and damaged cartilage and their precise roles have not been well defined. Extremely high amounts of leptin have been reported in obese individuals, which can be associated with osteoarthritis (OA) development. The aim of this study was to investigate whether BMPs could be induced in human primary chondrocytes during leptin-stimulated OA development and the underlying mechanism. We found that expression of BMP-2 mRNA, but not BMP-4, BMP-6, or BMP-7 mRNA, could be increased in human primary chondrocytes under leptin stimulation. Moreover, this BMP-2 induction was mediated through transcription factor-signal transducer and activator of transcription (STAT) 3 activation via JAK2-ERK1/2-induced Ser727-phosphorylation. Of note, histone deacetylases (HDACs) 3 and 4 were both involved in modulating leptin-induced BMP-2 mRNA expression through different pathways: HDAC3, but not HDAC4, associated with STAT3 to form a complex. Our results further demonstrated that the role of BMP-2 induction under leptin stimulation is to increase collagen II expression. The findings in this study provide new insights into the regulatory mechanism of BMP-2 induction in leptin-stimulated chondrocytes and suggest that BMP-2 may play a reparative role in regulating leptin-induced OA development.</p></div

Leptin induces BMP-2 expression in human primary chondrocytes.

<p>Cells were kept as controls or stimulated with leptin for 1, 4, 8, and 24 h, and the BMPs mRNA expressions (<i>A</i>) and protein secretion (<i>B</i>) were determined by real-time PCR and ELISA, respectively. Data in (<i>A</i>) and (<i>B</i>) are mean ± SEM from three independent experiments. *, <i>P</i> < 0.05 <i>vs</i>. untreated control cells.</p

STAT3-HDAC3 and HDAC4 are divergent pathways to mediate leptin-induced BMP-2 mRNA expression in human primary chondrocytes.

<p>Cells were kept as controls or treated with leptin for 1, 4, 8, and 24 h. (<i>A</i>) The associations of STAT3 with HDAC3 and HDAC4 were determined by an immunoprecipitation assay and Western blot and (<i>B</i>) the STAT3 acetylation was determined by Western blot. Results are representative of three independent experiments with similar results.</p