Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE)

Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.     

Haploinsufficiency of Akt1 Prolongs the Lifespan of Mice

There is increasing evidence that nutrient-sensing machinery is critically involved in the regulation of aging. The insulin/insulin-like growth factor-1 signaling pathway is the best-characterized pathway with an influence on longevity in a variety of organisms, ranging from yeast to rodents. Reduced expression of the receptor for this pathway has been reported to prolong the lifespan; however, the underlying mechanisms are largely unknown. Here we show that haploinsufficiency of Akt1 leads to an increase of the lifespan in mice. Akt1 ^+/– mice had a lower body weight than their littermates with less fat mass and normal glucose metabolism. Ribosomal biogenesis and the mitochondrial DNA content were significantly reduced in these mice, along with a decrease of oxidative stress. Consistent with the results obtained in mice, inhibition of Akt-1 promoted longevity in nematodes (Caenorhabditis elegans), whereas activation of Akt-1 shortened the lifespan. Inhibition of Akt-1 led to a decrease of ribosomal gene expression and the mitochondrial DNA content in both human cells and nematodes. Moreover, deletion of ribosomal gene expression resulted in a decrease of the mitochondrial DNA content and normalized the lifespan shortened by Akt-1 activation in nematodes. These results suggest that an increase of mitochondrial amount and energy expenditure associated with enhanced protein synthesis accelerates both aging and the onset of age-associated diseases.     

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo . Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli . The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.     

High expression of N-acetylglucosaminyltransferase V in favorable neuroblastomas: Involvement of its effect on apoptosis

Neuroblastoma (NBL), derived from the sympathetic precursor cells, is one of the most common pediatric solid tumors. The expression of N-acetylglucosaminyltransferase V and IX (GnT-V and GnT-IX) mRNA in 126 primary NBLs were quantitatively analyzed and higher expression levels of GnT-V were found to be associated with favorable stages (1, 2 and 4s). Conversely, the downregulation of GnT-V expression by small interfering RNA resulted in a decrease in the susceptibility to cell apoptosis induced by retinoic acid in NBL cells accompanied by morphological change. These results suggest that GnT-V is associated with prognosis by modulating the sensitivity of NBLs to apoptosis.     

Isolation and Characterization of a Novel Thraustochytrid-like Microorganism that Efficiently Produces Docosahexaenoic Acid

A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 °C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n−3) at 43% of the total fatty acids. It had a growth rate of 0.38 h⁻¹. The DHA production rate of 2.8 ± 0.7 g l⁻¹ day⁻¹ is the highest value reported for any microorganism. Received 7 October 2005; Revisions requested 7 October 2005; Revisions received 15 November 2005; Accepted 15 November 2005