Ras regulatory interactions: Novel targets for anti-cancer intervention?

Advances in the understanding of Ras oncoprotein function suggest novel points for anti-tumor intervention. First, upstream-acting guanine nucleotide exchange factors and SH2/SH3 domain-containing adaptor proteins that link Ras with growth factor receptor tyrosine kinases have recently been characterized. Second, work on downstream-acting Ras effector functions including the Ras GTPase-activating protein (p120^GAP) and the Raf kinase has revealed direct biochemical interactions that are functionally required for oncogenic Ras signalling. We summarize progress in these areas and discuss the potential for novel applications to anti-cancer chemotherapy.     

DBA/2J Genetic Background Exacerbates Spontaneous Lethal Seizures but Lessens Amyloid Deposition in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and is characterized by amyloid plaques, neurofibrillary tangles (NFTs) and neuronal dysfunction. Early onset AD (EOAD) is commonly caused by mutations in amyloid precursor protein (APP) or genes involved in the processing of APP including the presenilins (e.g. PSEN1 or PSEN2). In general, mouse models relevant to EOAD recapitulate amyloidosis, show only limited amounts of NFTs and neuronal cell dysfunction and low but significant levels of seizure susceptibility. To investigate the effect of genetic background on these phenotypes, we generated APP^swe and PSEN1^de9 transgenic mice on the seizure prone inbred strain background, DBA/2J. Previous studies show that the DBA/2J genetic background modifies plaque deposition in the presence of mutant APP but the impact of PSEN1^de9 has not been tested. Our study shows that DBA/2J.APP^swePSEN1^de9 mice are significantly more prone to premature lethality, likely to due to lethal seizures, compared to B6.APP^swePSEN1^de9 mice—70% of DBA/2J.APP^swePSEN1^de9 mice die between 2-3 months of age. Of the DBA/2J.APP^swePSEN1^de9 mice that survived to 6 months of age, plaque deposition was greatly reduced compared to age-matched B6.APP^swePSEN1^de9 mice. The reduction in plaque deposition appears to be independent of microglia numbers, reactive astrocytosis and complement C5 activity.     

Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time.     

Purification and properties of pyruvate kinase from Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M(r) of 250,000. It consists of four, apparently identical subunits each of M(r) 60,000. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.     

Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.