High-throughput retroviral tagging to identify components of specific signaling pathways in cancer

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.     

Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity

Neuropathy target esterase (NTE) is involved in neural development and is the target for neurodegeneration induced by selected organophosphorus pesticides and chemical warfare agents. We generated mice with disruptions in Nte, the gene encoding NTE. Nte(-/-) mice die after embryonic day 8, and Nte(+/-) mice have lower activity of Nte in the brain and higher mortality when exposed to the Nte-inhibiting compound ethyl octylphosphonofluoridate (EOPF) than do wild-type mice. Nte(+/-) and wild-type mice treated with 1 mg per kg of body weight of EOPF have elevated motor activity, showing that even minor reduction of Nte activity leads to hyperactivity. These studies show that genetic or chemical reduction of Nte activity results in a neurological phenotype of hyperactivity in mammals and indicate that EOPF toxicity occurs directly through inhibition of Nte without the requirement for Nte gain of function or aging.     

Mutations in the gene encoding peroxisomal alpha-methylacyl-CoA racemase cause adult-onset sensory motor neuropathy

Sensory motor neuropathy is associated with various inherited disorders including Charcot-Marie-Tooth disease, X-linked adrenoleukodystrophy/adrenomyeloneuropathy and Refsum disease. In the latter two, the neuropathy is thought to result from the accumulation of specific fatty acids. We describe here three patients with elevated plasma concentrations of pristanic acid (a branched-chain fatty acid) and C27-bile-acid intermediates. Two of the patients suffered from adult-onset sensory motor neuropathy. One patient also had pigmentary retinopathy, suggesting Refsum disease, whereas the other patient had upper motor neuron signs in the legs, suggesting adrenomyeloneuropathy. The third patient was a child without neuropathy. In all three patients we discovered a deficiency of alpha-methylacyl-CoA racemase (AMACR). This enzyme is responsible for the conversion of pristanoyl-CoA and C27-bile acyl-CoAs to their (S)-stereoisomers, which are the only stereoisomers that can be degraded via peroxisomal beta-oxidation. Sequence analysis of AMACR cDNA from the patients identified two different mutations that are likely to cause disease, based on analysis in Escherichia coli. Our findings have implications for the diagnosis of adult-onset neuropathies of unknown aetiology.     

Superoxide dismutase activity during the plasmodial life cycle ofPhysarum polycephalum

Summary Superoxide dismutase activity was slow throughout the cell cycle of surface cultures ofPhysarum polycephalum. This activity increased markedly when the organism was induced to spherulate. Glutathione (GSH) and hydrogen peroxide (H₂O₂) concentrations changed very little during the cell cycle. During spherulation GSH decreased; H₂O₂ and the cyanide-resistant respiration of plasmodial homogenates increased.     

Sclerotization and coloration of the insect cuticle

Summary Evidence indicating linkage between the processes of cuticle hardening and coloration are reviewed. Data and observations for the mutants black and ebony inDrosophila melanogaster are used to construct a possible model for the relationship between these developmental events.     

Influence of pinealectomy on corticotropin (ACTH)

Sensory deprivation produced by removing the eyes and olfactory bulbs in male rats allowed pinealectomy to markedly augment the post-adrenalectomy elevation of ACTH levels. Pineal removal or sensory deprivation separately did not have this effect. Thus, intact sensory input and an intact pineal gland are independently capable of restricting the post-adrenalectomy rise in ACTH levels.     

Occurrence,distribution and nature of neuropeptide Y in the rat pancreas

Summary Significant quantities of a newly discovered peptide, neuropeptide Y, were found in the rat pancreas, where they were localized to nerves in the exocrine parenchyma and around arterial and ductal structures. Although unaffected by surgical parasympathectomy, the periarterial and periductal nerves were abolished by chemical sympathectomy, suggesting that NPY is partially costored with sympathetic transmitters in nerve fibers.     

Timing, distribution, and abundance of kokanees spawning in a Lake Tahoe tributary

We counted kokanee spawners and carcasses every 1-7 days from mid-September through mid-November in 1991 and 1992 in Taylor Creek, a tributary to Lake Tahoe, California-Nevada. Less than 1% of the spawning run entered Taylor Creek before flow from Fallen Leaf Lake was increased on 2 October 1991; in 1992 the peak occurred on 30 September or 1 October after flows increased on 29 September. In both years spawners concentrated in the middle three of five stream reaches below the impassable Fallen Leaf Lake dam. From tab-and-recovery experiments, the average longevity of male spawners in the stream was 3.5 days in 1991 and 2.8 days in 1992, whereas the average female longevity was 2.0 days in 1991 and 2.3 days in 1992. Observed carcasses accounted for less than 10% of spawners counted, suggesting removal by scavengers or high predation on prespawners. An estimated 1928 males and 1309 females spawned in 1991, and 8021 males and 8712 females spawned in 1992. Our estimate of 3237 spawners in 1991 compared favorably to our estimate of 3520 ± 1474 prespawners staging in Lake Tahoe in mid-September. An index of kokanee abundance in Lake Tahoe has historically been based on 1-day surveys every 1 November since 1960; however, estimated total spawner abundance was 19 times higher than the annual index of 158 spawners in 1991, 141 times higher than the index count of 100 spawners in 1992. The index count and mean fork lengths of spawners (278 ± 10 mm [2 SE] for males, and 248 ± 3 mm for females) in 1991 and 1992 were the lowest on record.