Rapid Preparation of Thick Sections of Fleshy Plant Tissues

Methods are described for permanent micro-slide preparations of soft, large-celled plant tissues such as ripe fruit. Thick sections (200-800 [t) cut on a sliding microtome are aspirated in an aqueous killing agent; after fixing and washing, the sections are dehydrated and cleared in an alcohol-xylene series. Infiltration with 20, 30, and 40% solutions of mountant prior to mounting the sections is necessary to avoid too abrupt changes in the cleared tissues. Several staining methods have been successfully used for different purposes. The final preparations showed nearly perfect preservation of intact cells and intercellular spaces in their 3-dimension-al structure.     

OxLDL-induced gene expression patterns in CASMC are mimicked in apoE-/- mice aortas

Oxidized low density lipoprotein (oxLDL) contributes to the pathophysiology of atherosclerosis, partly by altering gene expression in vascular cells. Here, we show 221 genes differentially regulated by oxLDL in coronary artery smooth muscle cells (CASMC), using oligonucleotide microarrays. These genes were classified into 14 functional groups. A comparable gene expression pattern was detected in apoE(-/-) mice. OxLDL induced an oxidative stress response in CASMC, but not the unfolded protein response. OxLDL also caused CASMC death which was accompanied by increased expression of FasL, Bax, and p53 but was caspase-independent. This approach provides further insight into disease pathology and prognosis.     

Syk Tyrosine 317 Negatively Regulates Osteoclast Function via the Ubiquitin-Protein Isopeptide Ligase Activity of Cbl

Cytoskeletal organization of the osteoclast (OC), which is central to the capacity of the cell to resorb bone, is induced by occupancy of the αvβ3 integrin or the macrophage colony-stimulating factor (M-CSF) receptor c-Fms. In both circumstances, the tyrosine kinase Syk is an essential signaling intermediary. We demonstrate that Cbl negatively regulates OC function by interacting with Syk^Y317. Expression of nonphosphorylatable Syk^Y317F in primary Syk^−/− OCs enhances M-CSF- and αvβ3-induced phosphorylation of the cytoskeleton-organizing molecules, SLP76, Vav3, and PLCγ2, to levels greater than wild type, thereby accelerating the resorptive capacity of the cell. Syk^Y317 suppresses cytoskeletal organization and function while binding the ubiquitin-protein isopeptide ligase Cbl. Consequently, Syk^Y317F abolishes M-CSF- and integrin-stimulated Syk ubiquitination. Thus, Cbl/Syk^Y317 association negatively regulates OC function and therefore is essential for maintenance of skeletal homeostasis.OCs² are multinucleated cells generated by fusion of mononuclear progenitors of the monocyte/macrophage family under the aegis of M-CSF and receptor activator of nuclear factor κB ligand (RANKL) (1). Upon mineralized matrix recognition, the OC polarizes its fibrillar actin, eventuating in the formation of an acidified extracellular microenvironment that degrades bone. Failure to undergo this polarization event results in OC hypo-function and consequently in varying degrees of osteopetrosis (2).Integrins are transmembrane α/β heterodimers that mediate cell-cell and cell-matrix interactions and generate intracellular signals when occupied by ligands (3). The integrin, αvβ3, is expressed by OCs, and binding of this complex to bone is pivotal to the resorptive process (4).M-CSF recognizes its transmembrane receptor tyrosine kinase, c-Fms, and induces receptor autophosphorylation at seven tyrosine residues within the cytoplasmic domain (5). Several Src homology-2 domain-containing molecules are recruited to the phosphotyrosine residues upon M-CSF binding and initiate signaling cascades that lead to cytoskeletal organization, survival, and proliferation of OC lineage cells (5–7). Both the αvβ3 integrin and M-CSF are important regulators of OC actin remodeling (4, 6, 8).Syk is a 72-kDa nonreceptor tyrosine kinase, which mediates αvβ3- and c-Fms-induced OC cytoskeletal organization and function in a phosphorylation-dependent manner via a process involving activation of associated adaptor proteins, such as SLP-76 and Vav3 (9, 10). A number of Syk tyrosine residues undergo phosphorylation following engagement of the integrin and Fcγ receptor in immune (11) and mast cells (12). Three conserved tyrosine residues in the Syk linker region, namely Tyr³¹⁷, Tyr³⁴², and Tyr³⁴⁶, lie within consensus sequences for recognition by Src homology 2 domains, suggesting they transduce signals. Although phospho-Syk^Y342 and phospho-Syk^Y346 may serve as positive signaling regulators (12–14), phosphorylation of Syk^Y317 creates a binding site for c-Cbl, an E3 ubiquitin ligase proposed to prompt ubiquitination and subsequent degradation of Syk (15, 16). Hence, Syk^Y317 is a candidate negative regulatory site, but its role in OC function and/or differentiation is unknown.Cbl is a 120-kDa protein that is tyrosine-phosphorylated following activation by growth factors, cytokines, and integrins. It has two distinct but related activities, serving both as an adaptor protein (17, 18) and E3 ubiquitin ligase (19, 20). Cbl functions principally as an adaptor in OCs by participating in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton (18, 21). In other cell types, Cbl is also a negative regulator of receptor and nonreceptor tyrosine kinases, as it promotes their degradation (22). OCs and their precursors express c-Cbl and another family member Cbl-b that compensates for the absence of c-Cbl (23, 24). As combined deletion of both isoforms eventuates in early embryonic lethality (24), it is not clear if c-Cbl functions as an E3 ubiquitin ligase in OCs. We establish that c-Cbl, recognizing Syk^Y317, prompts the ubiquitination of the kinases thereby arresting activation of cytoskeleton-organizing molecules and thus OC function. The Cbl-Syk^Y317 complex is therefore important in maintenance of normal skeletal mass.     

The low abundance of clonally expanded mitochondrial DNA point mutations in aged substantia nigra neurons

Clonally expanded mitochondrial DNA (mtDNA) deletions accumulate with age in human substantia nigra (SN) and high levels cause respiratory chain deficiency. In other human tissues, mtDNA point mutations clonally expand with age. Here, the abundance of mtDNA point mutations within single SN neurons from aged controls was investigated. From 31 single cytochrome c oxidase normal SN neurons, only one clonally expanded mtDNA point mutation was identified, suggesting in these neurons mtDNA point mutations occur rarely, whereas mtDNA deletions are frequently observed. This contrasts observations in mitotic tissues and suggests that different forms of mtDNA maintenance may exist in these two cell types.     

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.     

Genome sequence of the Listia angolensis microsymbiont Microvirga lotononidis strain WSM3557T

Microvirga lotononidis is a recently described species of root-nodule bacteria that is an effective nitrogen- (N₂) fixing microsymbiont of the symbiotically specific African legume Listia angolensis (Welw. ex Bak.) B.-E. van Wyk & Boatwr. M. lotononidis possesses several properties that are unusual in root-nodule bacteria, including pigmentation and the ability to grow at temperatures of up to 45°C. Strain WSM3557^T is an aerobic, motile, Gram-negative, non-spore-forming rod isolated from a L. angolensis root nodule collected in Chipata, Zambia in 1963. This is the first report of a complete genome sequence for the genus Microvirga. Here we describe the features of Microvirga lotononidis strain WSM3557^T, together with genome sequence information and annotation. The 7,082,538 high-quality-draft genome is arranged in 18 scaffolds of 104 contigs, contains 6,956 protein-coding genes and 84 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.