The traditional practice of elevating the head in order to lower intracranial pressure (ICP) in head-injured patients has been challenged in recent years. Some investigators argue that patients with intracranial hypertension should be placed in a horizontal position, the rationale being that this will increase the cerebral perfusion pressure (CPP) and thereby improve cerebral blood flow (CBF). However, ICP is generally significantly higher when the patient is in the horizontal position. This study was undertaken to clarify the issue of optimal head position in the care of head-injured patients. The effect of 0 degree and 30 degrees head elevation on ICP, CPP, CBF, mean carotid pressure, and other cerebral and systemic physiological parameters was studied in 22 head-injured patients. The mean carotid pressure was significantly lower when the patient's head was elevated at 30 degrees than at 0 degrees (84.3 +/- 14.5 mm Hg vs. 89.5 +/- 14.6 mm Hg), as was the mean ICP (14.1 +/- 6.7 mm Hg vs. 19.7 +/- 8.3 mm Hg). There was no statistically significant change in CPP, CBF, cerebral metabolic rate of oxygen, arteriovenous difference of lactate, or cerebrovascular resistance associated with the change in head position. The data indicate that head elevation to 30 degrees significantly reduced ICP in the majority of the 22 patients without reducing CPP or CBF.

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 59 untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.[Keywords: DNA damage; translation; upstream ORF] Supplemental material is available at http://www.genesdev.org.

Early experience with continuous

The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.The proteins encoded by the myc gene family function as sequence-specific transcription factors that regulate target genes integral to the processes of cell proliferation, differentiation, and cell death (2, 9). Although many of the functions of the three major myc genes are overlapping, unique properties have been ascribed to individual Myc proteins. For example, each of the Myc proteins can restore the growth and proliferative defects of c-myc null fibroblasts and can promote apoptosis following growth factor deprivation (23,30). In addition, all three proteins can regulate known myc target genes (23). Unique roles for these proteins are supported by the following observations: during embryogenesis and in adult tissues, the expression patterns of c-, L-, and N-Myc are distinct (52); homozygous null c-or N-myc mice die early in development, whereas targeted disruption of both L-myc alleles is not lethal; and in some instances, L-myc displays distinct cell transformation and transcriptional properties (1, 38, 39).It is not surprising, given the role of the Myc family of proteins in proliferation and apoptosis, that the expression of these proteins is highly regulated at the levels of both transcription and translation. Indeed, deregulated Myc expression, through either of these mechanisms, has been associated with tumorigenesis (12,35,36,49,50).Previous studies have shown that the 5Ј untranslated regions (UTRs) of c-, L-, and N-myc encoded by exon 1 each contain a complex RNA structural element known as an internal ribosome entry segment (IRES). Consequently, synthesis of the Myc family proteins can occur by the process of internal ribosome entry (18,19,28,45). In this mechanism of translation initi...

A method is described for the detection of certain nucleotide modifications adjacent to the 5' 7-methyl guanosine cap of mRNAs from individual genes. The method quantitatively measures the relative abundance of 2'-O-methyl and N6,2'-O-dimethyladenosine, two of the most common modifications. In order to identify and quantitatify the amounts of N6,2'-O-dimethyladenosine, a novel method for the synthesis of modified adenosine phosphoramidites was developed. This method is a one step synthesis and the product can directly be used for the production of N6,2'-O-dimethyladenosine containing RNA oligonucleotides. The nature of the cap-adjacent nucleotides were shown to be characteristic for mRNAs from individual genes transcribed in liver and testis.

The taxanes are a group of polycyclic diterpenes produced by various species of yew. The potent anticancer drug paclitaxel (marketed as Taxol) is the commercially most important taxane with annual sales in 2000 exceeding 1.6 billion dollars. Paclitaxel is currently obtained either by direct extraction from yew trees or by the extraction of the precursor 10-deactilbaccatin III, which is then converted to paclitaxel by semi-synthesis. Apart from cost, one of the main draw backs to taxol in cancer treatment is the development of resistance by tumours, commonly due to the expression of ABC transporter efflux pumps which remove the drug from the target cell. A number of natural taxanes and semisynthetic derivates, have recently been shown to act as potent inhibitors of ABC transport proteins. These compounds have no effect upon microtubule polymerization (the normal target of paclitaxel), but have the ability to restore drug sensitivity when given in combination with paclitaxel to resistant cell lines. In work to be described elsewhere, we sort to carry out a structure function analysis of the ability of novel oxidised taxanes to act as ABC transporter inhibitors. For this study 100 mg or more of taxadiene [taxa-4(5),11(12)-diene], the first taxane in the paclitaxel pathway, was required as starting material from which to synthesize these compounds. Taxadiene is synthesised directly from geranylgeranyl diphosphate (GGPP), which is found in most plant tissues where it serves as a common precursor for many metabolites. The synthesis and use of GGDP are tightly regulated in most vegetative organs, however, in tomato fruit it is used almost exclusively for the production of coloured carotenoids which accumulate to high levels in the plastid as lycopene crystals. Expressing taxadiene synthase in a yellow-fruited tomato line that lacks the ability to utilise GGPP for carotenoid synthesis allowed GGPP normally utilised for making carotenoids to be re-routed for the production of taxadiene, allowing the facile extraction of 160 mg of highly pure taxadiene from 1 kg of freeze dried fruit.

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

Despite the wide synthetic potential of diazo compounds (XÀH insertion, ylide formation, cyclopropanation, cycloaddition etc.), [1] concerns over the hazards associated with their preparation, isolation, and use have hindered their full exploitation in both academic and industrial laboratories. A few diazo compounds are commercially available (e.g. ethyl and butyl diazoacetate, TMS-diazomethane and diazodimedone), [2] but safe and convenient access to a wider range of useful functionalized diazo species is still desirable.[3] Diazo transfer can be used to access a-diazocarbonyls, but this only partially addresses the safety concerns associated with the diazo species, as the use of equally hazardous azidebased diazo-transfer reagents is still required.[4] Ideally, it would be beneficial if the diazo species could be generated and consumed in situ so that handling of the hazardous diazo compound is avoided altogether.Recent work by Ley, [5] Jamison, [6] Kappe, [7] and others [4,[8][9][10] has shown that highly reactive diazo and azido compounds can be used in lab-scale continuous-flow reactors to achieve a number of very useful synthetic transformations, and indeed work from our own laboratory has shown that ethyl diazoacetate can be used in-flow to access b-keto esters.[11] We therefore wondered if it was possible to actually generate a-diazocarbonyl compounds under flow conditions and then use these materials directly in further synthetic manipulations, thus minimizing exposure to any potentially hazardous material. In effect, could we develop a continuous-flow diazo generator and then demonstrate its use to prepare a range of useful a-alkoxy (3 a-i) and aamino acid (4 a-i) derivatives through O À H and N À H insertion (Scheme 1)?At the outset we were aware that in order to provide an acceptable solution to the problem, we needed to identify a way to access the diazo compounds of interest (2 a-i) from starting materials that showed an acceptable safety profile, that is, the precursor molecules and reagents should be safer to prepare and handle than the diazocarbonyl compounds being produced. Of the methods available for the generation of a-diazocarbonyl compounds, we were particularly attracted to the Bamford-Stevens reaction as it uses readily accessible arylsulfonylhydrazones (e.g., 1 a-i) as starting materials, with the corresponding diazocarbonyls being generated upon exposure to relatively weak base at moderate reaction temperatures. [12][13][14] Thermal stability studies (DSC and TGA) were conducted on the tosylhydrazone 1 b and its corresponding methyl diazoester 2 b [15,16] in order to determine if a safe window of operation could be identified for the continuous-flow process (see the Supporting Information). The results clearly show that the rate of initial mass loss from diazoester 2 b peaks at 125 8C, which corresponds to a significant exotherm. In comparison tosylhydrazone 1 b has a rate of mass loss which peaks at 221 8C, indicating that it is substantially more thermally stable.[15] We therefore co...