This Week in PNAS
APPLIED PHYSICAL SCIENCES
Gold nanorods for imaging
Haifeng Wang et al. use the bright two-photon luminescence of gold nanorods to turn these rods into probes for in vitro and in vivo imaging. Wang et al. determined that one gold nanorod has a two-photon luminescence signal 58 times brighter than that of a two-photon fluorescence signal from a single rhodamine molecule. The brightness is due to a plasmon-enhanced two-photon absorption cross section. Wang et al. excited and imaged prepared nanorods with an 830-nm laser-scanning microscope, using this setup to image the blood vessels of a live mouse after nanorod injection into the tail vein. Broadband emissions were detected from individual nanorods found to be flowing in mouse ear blood vessels. The rods were visible 5 min after the injection and dissipated fully by 30 min. The nanorods' brightness depended on the incident polarization, providing a way to calculate the rods' orientation in a given sample. The authors suggest that the rods' brightness and the ability to measure the nanorods' orientation may advance biological imaging. — P.D.
“In vitro and in vivo two-photon luminescence imaging of single gold nanorods” by Haifeng Wang, Terry B. Huff, Daniel A. Zweifel, Wei He, Philip S. Low, Alexander Wei, and Ji-Xin Cheng (see pages 15752–15756)
BIOCHEMISTRY
Lack of receptor N-glycosylation linked to emphysema
Xiangchun Wang et al. report that disrupting core fucosylation of the TGF-β1 receptor causes emphysema-like disorders in mice. Previous research has implicated the up-regulation of matrix metalloproteinases (MMPs) in the induction of emphysema, and the TGF-β1 receptor-mediated signaling pathway is known to suppress the expression of MMPs. Mutations in fucose modifications of proteins have been linked to a number of congenital glycosylation disorders, as well as to hepatocellular carcinomas and liver cirrhosis. In mammals, the Fut8 gene catalyzes fucose modifications on glycoproteins, and Wang et al. engineered mice lacking this gene. Although the Fut8 -/- mutant animals were born healthy, 70% died within 3 days, and the survivors had severe growth retardation. The Fut8 -/- mice displayed widespread air space enlargement and dilated alveolar ducts. Increased levels of MMPs were detected in Fut8 -/- mice. Embryonic fibroblasts derived from Fut8 -/- mice showed a decreased response to TGF-β1 stimulation, with the binding ability of the receptor reduced significantly compared with wild type. Intraperitoneal injection of exogenous TGF-β1 rescued the null mice from the emphysema-like phenotype. — F.A.
“Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice” by Xiangchun Wang, Shinya Inoue, Jianguo Gu, Eiji Miyoshi, Katsuhisa Noda, Wenzhe Li, Yoko Mizuno-Horikawa, Miyako Nakano, Michio Asahi, Motoko Takahashi, Naofumi Uozumi, Shinji Ihara, Seung Ho Lee, Yoshitaka Ikeda, Yukihiro Yamaguchi, Yoshiya Aze, Yoshiaki Tomiyama, Junichi Fujii, Keiichiro Suzuki, Akihiro Kondo, Steven D. Shapiro, Carlos Lopez-Otin, Tomoyuki Kuwaki, Masaru Okabe, Koichi Honke, and Naoyuki Taniguchi (see pages 15791–15796)
BIOPHYSICS
Proteins slide, hop, and jump on DNA
Darren Gowers et al. have devised a method to analyze short-distance protein motion on DNA. Proteins that act at specific DNA sequences first bind DNA randomly and then transfer to the target site by facilitated diffusion. The nature of short-range protein translocations on DNA, at distances <100 bp, have not been well understood. Gowers et al. employed the BbvCI restriction enzyme and constructed radioactively labeled plasmids with two BbvCI cleavage sites separated by 30–75 bp. Restriction sites on the plasmids were oriented either head-to-tail or head-to-head. BbvCI displayed a high level of processivity on the plasmid with repeated sites and a lower level on the substrate with inverted sites when the sites were 30 bp apart. This difference in processivities suggests that BbvCI can slide along the DNA, remaining in continuous contact with the nucleotide chain. At a 75-bp separation, however, the enzyme displayed a similar processivity for both head-to-head and head-to-tail sites. This similarity may be due to BbvCI hopping or jumping between DNA sites by dissociating from the initial target site and reassociating at the second cleavage site. — F.A.
“Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA” by Darren M. Gowers, Geoffrey G. Wilson, and Stephen E. Halford (see pages 15883–15888)
GENETICS
Megacloning of bacterial DNA
Mitsuhiro Itaya et al. have developed a method to transfer whole genomes from one bacterial species into another. Most DNA transfer vectors cannot handle a large number of genes at one time. To increase the genomic size for DNA cloning, Itaya et al. utilized the BGM cloning vector derived from the 4.2-Mb genome of Bacillus subtilis bacteria. For target DNA, the authors used the 3.5-Mb genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis. The Synechocystis genome was divided into four regions of approximately equal size and guided into the BGM vector by simultaneous homologous recombination at landing pad sequences, small flanking DNAs integrated at the BGM cloning locus. The two landing pad sequence sites slid to accommodate the adjacent target DNA by the process of inchworm elongation. Instead of repeating inchworm elongation for each of the four Synechocystis regions, the authors progressively transferred the existing intermediate BGMs that they had already obtained. Two RNA operon genes were deleted from the Synechocystis genome in order to successfully clone it into the B. subtilis genome. The resultant bacteria, with a composite genome 7.7 Mb in size, grew only in the B. subtilis culture medium in which the cloning procedures were carried out. — F.A.
“Combining two genomes in one cell: Stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome” by Mitsuhiro Itaya, Kenji Tsuge, Maki Koizumi, and Kyoko Fujita (see pages 15971–15976)
MICROBIOLOGY
Yersinia exerts effect through Toll-like receptor
Andreas Sing et al. show that the N-terminal region of the Yersinia LcrV protein induces IL-10 production in macrophages via a Toll-like receptor 2 (TLR2) pathway. Previous research has shown that LcrV, the virulence antigen of the plague bacteria Yersinia, functions as an immunomodulator, but the mechanism for this activity was not known. Sing et al. sequenced the lcrV genes of Yersinia to detect amino acid polymorphisms in the N-terminal region. Synthetic peptides and recombinant truncated LcrV proteins strongly induced gene expression in cells transiently transfected with TLR2. A mutation in the N-terminus abolished LcrV's ability to activate gene expression. Wild-type mice infected with the mutant Yersinia survived longer than mice infected with wild-type Yersinia. In TLR2- or IL-10-null mice, survival rates and bacterial loads did not significantly differ between those infected with wild-type versus mutant Yersinia. In addition, IL-10 levels in the spleen were lower in wild-type mice infected with the mutant bacterial strain than mice infected with wild-type bacteria. — F.A.
“A hypervariable N-terminal region of Yersinia LcrV determines Toll-like receptor 2-mediated IL-10 induction and mouse virulence” by Andreas Sing, Dagmar Reithmeier-Rost, Kaisa Granfors, Jim Hill, Andreas Roggenkamp, and Jürgen Heesemann (see pages 16049–16054)
Figures and Tables
In vivo imaging of single gold nanorods (red) in mouse ear blood vessels.
Therapeutic administration of TGF-β1 rescues emphysema-like changes in Fut8 -/- mice.
Protein transfer on DNA repeated sites by sliding (Left) and on inverted sites by jumping (Right).
Structure of the megacloned Synechocystis genome in the B. subtilis genome.
Cells infected with mutant Yersinia show similar phagocytosis resistance as wild type.
