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[en] In this chapter we discussed methods that can be used for the sensitive detection and quantitation of differentially or alternatively spliced mRNAs as well as mRNAs of low abundance. Although mechanisms responsible for splicing (and differential splicing in particular) have not been fully determined, many RNAs derived from a variety of genes have been observed to undergo the process. The impact of splicing with regard to the expanded potential of gene expression emphasizes the usefulness of the solution hybridization-nuclease digestion technique described here, compared to Northern blot analysis. The use of radiolabeled cRNA(s) provides for an assay of both high specificity and high sensitivity. While end-labeled cDNA probes can be used, they do not have the sensitivity inherent in the assay performed with uniformly radiolabeled cRNAs. If multiple mRNAs are derived from a single gene as a result of differential or alternative precursor RNA splicing, however, the results with a cRNA probe may initially appear to be quite complicated, and end-labeled cDNAs may yield more easily interpretable results. Nonetheless, both types of probes are useful in the context of gene expression analysis, and it is clear that for routine purposes of quantitation cRNA probes in solution hybridization-nuclease protection assays are clearly more desirable than RNA blot analyses due to their truly quantitative nature as well as ease of assay
[en] Complete text of publication follows. Genomic instability has been demonstrated in the progeny of irradiated cells and unirradiated bystander cells. Bystander responses are thought to depend on the activation of cellular communication processes. In this study we examine one such mediator of cellular communication, the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) TNF-α is known to increase in expression following ionizing radiation (IR) exposure. Upon binding to its cellular receptors, TNF-α initiates a signaling cascade mediated by reactive oxygen species (ROS) that can activate sequestered NF-κB, thus initiating a pro-inflammatory and antiapoptotic pathway. NF-κB can in turn upregulate TNF-α expression, which when secreted can induce subsequent autocrine and paracrine stimulation of TNF-α and NF-κB. We speculate that this increase in TNF-α signaling and concomitant ROS generation has a mechanistic role in the initiation of genomic instability and a potential involvement in producing bystander responses. Genomic instability is induced by IR in a non-dose-dependent manner. Previous investigation by our group using primary human vascular endothelial cells has shown that both low (0.1 Gy) and high (2 Gy) doses of IR raise levels of secreted TNF-α in a non-linear manner, that both immediate genetic damage and delayed chromosomal instability can be induced at similar levels following treatment with either 0.1-10 ng/mL TNF-α or 0.1 or 2 Gy IR, and that this immediate damage was abrogated by pre-incubation with antioxidants. The current study is therefore focused on the mechanism responsible for this TNF-α-induced instability, and whether TNF-α is a signaling mediator of bystander-induced responses. TNF-α suppressors are added to either directly irradiated or bystander cell cultures exposed to low or high doses of low-LET radiation, and the results are compared to cells pre-treated with antioxidants. Cellular damage is assessed by cell survival, the comet assay, formation of damage-induced foci, and presence of delayed chromosomal instability. Preliminary results indicate that: 1) suppressing TNF-a prevents immediate genetic damage in directly irradiated cells, similar to antioxidant treatment, 2) antioxidants but not TNF-a suppression can abrogate delayed chromosomal instability in directly irradiated cells, and 3) the suppression of either TNF-α or nitric oxide in bystander cells increases survival and protects against immediate genetic damage after exposure to medium from cells irradiated with 2 Gy.
[en] The synthesis of a new acetylene analogue of the C-terminal heptapeptide segment of cholecystokinin (CCK) in which the Met28 and Met 31 residues are replaced by two acetylene precursors of norleucine (Nle), L-2-amino-4hexynoic acid (Aha), is described. Reductive tritiation of this acetylene heptapeptide Boc:Ahasup(28,31):-CCKsub(27,33) led to the labelled :3H:-Boc-:Nlesup(28,31):-CCKsub(27-33) which displays a specific activity of about 150 Ci/mmol. According to its full biological potency, this CCK analogue can be used for various biological assays including binding studies. (author)
[en] When the cellular glutathione content is reduced, adding oxygen (130 μmol dm-3) 7 ms after irradiation of hypoxic cells increases the radiosensitivity (factor approx. 1.25), whereas it has much less effect in normal cells. (author)
[en] Amyloid formation and aberrant protein aggregation have been implicated in more than 15 different human diseases and an even wider range of proteins form amyloid in vitro. From a structural perspective the proteins which form amyloid can be divided into two classes: those which adopt a compact globular fold and must presumably at least partially unfold to form amyloid and those which are unstructured in their monomeric state. Important examples of the latter include the Aβ peptide of Alzheimer's disease, atrial natriuretic factor, calcitonin, pro-calcitonin, islet amyloid polypeptide (IAPP, amylin), α-synuclein and the medin polypeptide. The kinetics of amyloid assembly are complex and typically involve a lag phase during which little or no fibril material is formed, followed by a rapid growth stage leading to the β-sheet-rich amyloid structure. Increasing evidence suggests that some natively unfolded polypeptides populate a helical intermediate during the lag phase. We propose a model in which early oligomerization is linked to helix formation and is promoted by helix–helix association. Recent work has highlighted the potential importance of polypeptide membrane interactions in amyloid formation and helical intermediates appear to play an important role here as well. Characterization of helical intermediates is experimentally challenging but new spectroscopic techniques are emerging which hold considerable promise and even have the potential to provide residue specific information