Inlet hydrogenation gas chromatography to predict mass % linear paraffin content.

The light condensate fraction obtained from the low temperature Fischer-Tropsch (LT-FT) process is very complex and it is processed further by hydrotreating to produce hydrocarbon products that can be sold as final products. The mass% linear paraffins in some of the final paraffin products is listed as a required specification. Usually gas chromatography equipped with a flame ionisation detector (GC-FID) is used for the analysis of the condensate feeds to estimate the mass% linear paraffins that can be expected in the final products after commercial hydrogenation. This is an important parameter used in the blending of suitable condensate feeds. Due to the complexity of the condensate feeds, significant peak overlap occurs in the GC-FID analysis, making it difficult to accurately estimate the mass% linear paraffin content that will be obtained in the hydrogenated products. Inlet hydrogenation GC-FID analysis simplifies the prediction of the mass% linear content that can be expected in the paraffin product fractions from the analysis of a plant feed since the feed is hydrogenated in the GC inlet before GC-FID analysis. The results from this study showed that sufficient hydrogenation without significant peak tailing can be obtained in the GC inlet when using the appropriate mass and particle size Pd/Al2O3 catalyst with the optimum bed height. Inlet hydrogenation GC-FID analysis simplifies the prediction of the mass% linear content that can be expected in the paraffin product fractions. The method can be implemented on routine GC-FID instrumentation by simply installing an inlet liner containing an appropriate catalyst, that could be re-used at least 20 times, and avoids the purchasing of additional instrumentation and complex data processing and is suitable for commercial process control.

An analysis of results from 305 compounds tested with the yeast RAD54-GFP genotoxicity assay (GreenScreen GC)-including relative predictivity of regulatory tests and rodent carcinogenesis and performance with autofluorescent and coloured compounds.

Data from 305 non-proprietary compounds tested using the yeast RAD54-GFP (Green Fluorescent Protein) assay, GreenScreen GC, are presented, together with a detailed comparison with results from in vitro and in vivo genotoxicity tests and rodent carcinogenesis. In addition, observations on reproducibility and the performance of the test with autofluorescent and coloured compounds are described. Like the Ames test, the GreenScreen assay is shown to exhibit high specificity (82%), meaning that compounds with positive results are very likely to be genotoxic carcinogens. This is in contrast to mammalian cell tests established for use in regulatory testing that provide disappointingly low specificity and the inevitable generation of confounding false positive data. The analysis confirmed the observations of earlier studies, showing that a combination of an Ames test (or surrogate) with the yeast test provides high specificity as well as high sensitivity in the identification of rodent carcinogens.

An assessment of the utility of the yeast GreenScreen assay in pharmaceutical screening.

In this paper we describe an initial reproducibility study of 12 proprietary compounds followed by the assessment of 51 marketed pharmaceuticals and, lastly, a summary of the data so far from 2698 proprietary compounds from the Johnson & Johnson (J&J) compound library, in the yeast GreenScreen assay (GSA). In this assay, a reporter system in the yeast cells employs the DNA damage inducible promoter of the RAD54 gene, fused to the extremely stable green fluorescent protein (GFP). The assay proved to be very robust, the Excel templates provided by Gentronix with the assay interfaced well with in-house J&J systems with little adaptation, the assay was very rapid to perform and used very little compound. The results confirm previous work which suggests that the yeast GSA detects different classes of genotoxic compounds to the Ames assay and as a result can help screen out important genotoxic compounds at the pre-regulatory test phase that are missed by Ames-test-based screens alone. A combination of SAR evaluation of genotoxicity plus an Ames-test-based screen and the GSA provides a powerful pre-regulatory test battery to aid in the selection of successful drug candidates.

Dieulafoy's lesion: a case series study.

AIM: Dieulafoy's lesion (DL) accounts for 1-5.8% of cases of acute upper gastrointestinal bleeding (GIB). Its mortality is high, approaching 20%, despite recent advances in endoscopic therapy. We aimed to report our experience in the treatment of DL. METHODS: A retrospective case study of all patients with DL between January 1993 and January 2003 was done. Characteristics, treatment methods, success rates and 30-d mortality of the patients were analyzed. RESULTS: Thirty-six patients were noted to have DL in the study period. Thirty-three records were available for assessment in which 35 DL were identified. The median age of the patients was 67 years with male to female ratio of 5.6:1. Significant comorbidities existed in 69% of the patients. Eighty-nine percent of the DL was found at first endoscopy, three DL at laparotomy. Significant coexistent endoscopic findings existed in 23%. Hemostasis was achieved in 88% by using adrenaline injection, or in combination with heater probe application at first endoscopy. Four cases had re-bleeding, all were successfully treated endoscopically. The 30-d mortality rate was 23%. CONCLUSION: Successful endoscopic hemostasis could be achieved in 100% of cases of DL. The overall mortality may still remain high, mainly due to the comorbidities and age of these patients.

Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP genotoxicity assay.

The yeast (Saccharomyces cerevisiae) RAD54-GFP DNA repair reporter assay (GreenScreen assay, GSA) can be used for early genotoxicity screening in drug discovery. During the initial validation of this preregulatory assay, a subset of known genotoxic compounds that did not give reproducibly clear positive GSA results was identified. Cell permeability, inherent drug resistance mechanisms, metabolic activation and compound solubility were identified as possible barriers to the detection of specific compounds. In this study three types of modification to the existing assay protocol were explored in order to address these possibilities: (i) modification of the reporter host strain by deletion of genes involved in cell wall integrity or with products functioning as efflux pumps (PDR5, ERG6, SNQ2, YOR1); (ii) expression in the host yeast of human phase I metabolic activation genes and (iii) variation in the test solvent system for compounds with poor aqueous solubility. The modifications described and the assay results presented show how the assay may be tailored to suit specific classes of test compound in a more analytical mode. Improvements in assay sensitivity were seen in the detection of some genotoxins using yeast cell wall mutants and those expressing human cytochrome P450 genes.

Results of a technology demonstration project to compare rapid aquatic toxicity screening tests in the analysis of industrial effluents.

The results of a 'BioWise' demonstration project to assess the comparative sensitivity and practicality of seven new assays for the direct assessment of ecotoxicity in industrial effluents are presented. In addition the aim of the project was to validate the results of the new assays against benchmark data generated from non-proprietary, rapid, microplate screening assays using the regulatory species; freshwater crustacean Daphnia magna and green algae Selenastrum capricornutum, chosen in view of their environmental relevance. The new commercial test assays were: Daphnia magna, Selenastrum capricornutum and Thamnocephalus platyurus Toxkits supplied by Vickers Laboratories Ltd, containing dormant, immobilised life stages of the test species; GreenScreen EM, a yeast based assay for genotoxicity and general acute toxicity supplied by Gentronix Ltd; and CellSense a mediated, amperometric whole cell biosensor based on immobilised activated sludge and E. coli. 38 effluent samples supplied by members of SOCSA (Specialised Organic Chemicals Sector Association) were examined over a period of 13 months, in the project co-ordinated by the AstraZeneca Brixham Environmental Laboratory, and part funded by BioWise via the UK Government Department of Trade and Industry.

Unequal sister chromatid exchange in the rDNA array of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae the nucleolar organiser region (NOR) is located on chromosome XII. It contains 100-200 copies of rDNA--a minimum of 20 rDNA genes in tandem--and is termed the RDN locus. Yeast cells may exist in either haploid or diploid form. There are two forms of life cycle: haploid and diploid cells double by mitosis, and diploid cells are reduced to the haploid state by meiosis. Diploid cells have two homologous chromosomes for each of the 16 chromosomes. They are usually of the same size. However, in this study it is shown that homologous chromosomes XII can become different in size due to unequal sister chromatid exchange during mitosis in 'old' cells.

The GreenScreen genotoxicity assay: a screening validation programme.

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.

Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species.

The genes encoding GDP-mannose pyrophosphorylase from Saccharomyces cerevisiae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the control of the tightly regulated promoters of MET3 and CaMET3 respectively. Northern analysis showed that the addition of methionine effectively blocks the transcription of pMET3-SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants. Methionine-mediated repression of CaSRB1 caused loss of viability in C. albicans, demonstrating that, as in S. cerevisiae, the gene is essential for growth. Depletion of GDP-mannose pyrophosphorylase had a highly pleiotropic effect in the two yeasts. The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and bud's site selection, clumping and flocculation, as well as increased sensitivity to a wide range of antifungal drugs and cell wall inhibitors, and impaired hyphal switching ability. These phenotypes resulted from defects in glycosylation, as demonstrated by reduced affinity for Alcian blue and sensitivity to hygromycin B. Our results provide new information about the roles of protein glycosylation in yeast and, in particular, the steps that require GDP-mannose in the fungal pathogen C. albicans.

Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells.

PURPOSE: To optimise the high efficiency, non-viral transfer of DNA to retinal pigment epithelial (RPE) cells in vitro. METHODS: A mammalian expression vector (pcDNA3.1) containing a firefly luciferase (luc) cDNA was used to transfect RPE cells using different chemical methods; calcium phosphate, DEAE-dextran and, liposomes-based transfection techniques. Transfection was optimised for both dose and time of exposure. The efficiency of gene transfer and cytotoxicity was measured 48 hours post-transfection using luciferase and MTT assays, respectively. The percentage of transfected cells (using optimal conditions) was determined with a construct expressing a jellyfish green fluorescent protein (GFP) using flow cytometery. RESULTS: Calcium phosphate and DEAE-dextran techniques failed to transfect the vector and led to high cytotoxicity. Liposomes-based methods successfully transferred the vector to RPE cells, but the efficiency varied for different liposomes; Tfx-50 > Lipofectin > Lipofectamine > Cellfectin > DMRIE-C. No significant cytotoxicity was observed with any of the liposome treatments. Optimal transfection was achieved with Tfx-50 at a 3:1 ratio of DNA:liposome; between 12-15% of cells being transfected. CONCLUSIONS: Efficient and non-toxic transfer of functional genes into primary RPE cells in vitro can be successfuly achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.

Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances.

Yeast strains transformed with high copy number plasmids carrying the gene encoding a green fluorescent protein optimised for yeast (yEGFP3) under the control of the RAD54 or RNR2 promoter were used to investigate the activity of potentially DNA-damaging substances. The assays were performed on 96-well microtitre plates in the presence of different concentrations of the test substances. The synthesis of GFP protein was measured through the fluorescence signal and cell growth was monitored by absorption. Here, we demonstrate that this system can be used as a biosensor to assess the genotoxic potential of drugs and other chemical substances. The use of microtitre plates will enable full automation of the system and allows the inclusion of internal reference standards in each assay.

Regulation of tyrosinase expression and activity in cultured human retinal pigment epithelial cells.

The purpose of this study was to investigate the regulation of tyrosinase gene expression and activity in cultured human retinal pigment epithelial (RPE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplified and cloned into a modified mammalian expression vector (pcDNA3.1) upstream of a firefly luciferase (Luc) cDNA and was designated 'pcDNA3.1-Ty.prom.Luc'. The plasmid was co-transfected into RPE cells with a second mammalian expression plasmid (pRL-TK) containing a herpes simplex virus thymidine kinase promoter region upstream of Renilla Luc in a protocol designated the 'dual luciferase assay' (DLA). After co-transfection, cells were treated with a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, alpha-melanocyte stimulating hormone, verapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and L-tyrosine. The expression of tyrosinase promoter and enzymatic activities were determined 48 hr post-transfection using the DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not be detected in RPE cells with any of the treatments. Tyrosinase promoter activity was significantly up-regulated in RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, it appears that RPE cells in culture lack a post-transcriptional and/or translational modification point(s), which are necessary for tyrosinase enzymic activity.

Replicative ageing in the fission yeast Schizosaccharomyces pombe.

Saccharomyces cerevisiae has been widely used as a model organism in studies of replicative ageing and senescence. The relevance of these studies to ageing in other organisms has, however, been questioned, since this yeast divides by budding rather than fission, the more common pattern in higher organisms. Here we report that, contrary to popular belief, the fission yeast Schizosaccharomyces pombe also undergoes replicative senescence and in a manner superficially analogous to budding yeast. These experiments provide the first evidence of age asymmetry in cell fission and are consistent with the hypothesis of Jazwinski, that asymmetric division underlies culture immortality. Given their evolutionary divergence, comparison of the ageing determinants in fission and budding yeasts may help identify common mechanisms of the ageing process.

Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae.

Deletion via PCR-mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading-frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short-flanking homology (SFH) or a long-flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non-growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non-essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap-repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Anti-neutrophil cytoplasmic autoantibodies (ANCA) to bactericidal/permeability-increasing (BPI) protein recognize the carboxyl terminal domain.

OBJECTIVES: to identify the region of bactericidal/permeability-increasing protein (BPI) recognized by anti-BPI ANCA. METHODS: sera from 140 patients with a variety of clinical diagnoses (20 systemic vasculitis, 12 cystic fibrosis, 22 bronchiectasis/chronic obstructive airways disease, three diabetes mellitus, 13 chronic renal failure, 12 primary sclerosing cholangitis, eight ulcerative colitis, three Crohn's disease, seven cancer, and 40 other or unknown diagnoses) known to be reactive against native (nBPI), were screened by solid phase enzyme linked immunosorbent assay (ELISA) against a panel of recombinant fusion proteins; holo BPI (rBPI), recombinant lipopolysaccharide binding protein (rLBP), an N-terminal fragment of rBPI (rBPI21 ) and 'fusion' proteins containing the C- or N-terminal ends of BPI spliced with N-or C-ends of LBP, respectively. RESULTS: a strong correlation was seen between the degree of reactivity to rBPI and the BPI C-terminal fusion protein, r=0.69, P < 0.001, as well as between nBPI and rBPI protein, r=0.55, P < 0.001, but not between nBPI and the N-terminal region of BPI (rBPI21), or proteins containing only the N-terminal fragment. Binding to proteins containing the BPI C-terminus was confirmed to be specific by fluid phase inhibition ELISA and Western blot analyses. CONCLUSIONS: together these data suggest that circulating autoantibodies to BPI from patients with different diseases recognize the C-terminal region of BPI.

Application of an in vitro model to evaluate bioadhesion of fibroblasts and epithelial cells to two different dressings.

The cellular component of a healing wound consists of many cell types and the environment in which these cells grow is important to the rate and quality of healing which can be influenced by the type of dressing used. The most commonly used dressings are traditional gauze-type dressings. In many cases these dressings may adhere to the wound surface, and subsequent removal is often traumatic, causing pain and tissue reinjury. Some modern gelling dressings have been developed to overcome this adherence problem. In order to evaluate in more detail cell-dressing interactions, an in vitro model has been developed utilising wound fibroblasts and epithelial cells. Quantitative evaluation of adherence of cells cultured with a traditional gauze or a new gelling dressing has been undertaken using radiolabel and manual counting techniques. Scanning electron microscopy has been used to visualise the cells adherent to dressings allowing evaluation of their adhesion-morphology. The results show differential attachment of cells to viscose and gelling fibres of the dressings; considerably reduced cell adhesion to the gelling fibre was evident, and it was apparent that cells adhered predominantly to the viscose component of the dressing. This model can be used to investigate and compare the adhesion of cells to different dressings and their components.

Clinical correlation of a skin antisepsis model.

The use of pigskin as a test substrate for evaluating topical antimicrobial activity has been developed. Simulated handwashing protocols with this in vitro model in parallel with in vivo studies have been evaluated, based on an ASTM method for the clinical evaluation of a healthcare personnel handwash. Using Serratia marcescens as the test organism, similar log reductions were observed using the in vitro model when compared to in vivo efficacy. Results suggest that this model can be used as a reliable indicator of antiseptic efficacy on the skin. The use of sterilized skin simplifies the use of this model for both efficacy and skin-pathogen interaction studies.

Adsorption of serum-derived proteins by primary dressings: implications for dressing adhesion to wounds.

Using an in vitro immunolocalization technique, an exploratory study was carried out into the serum-derived protein adsorption capacity and the cell adherence of a traditional gauze dressing versus a new gelling fibre gauze dressing. We found that the traditional gauze dressing adsorbed protein more readily than the new dressing. The findings indicate that reduced binding of serum proteins to the surface of the gelling fibre dressing may help reduce the adherence characteristics for this type of dressing, minimising trauma and possibly reducing the acute pain experienced during dressing changes.

Development of a green fluorescent protein reporter for a yeast genotoxicity biosensor.

A reporter system, constructed for a laboratory screen for new genes involved in DNA repair in the brewer's yeast Saccharomyces cerevisiae, has been developed for use in a genotoxicity biosensor. The strain produces green fluorescent protein (yEGFP) when DNA damage has occurred. yEGFP is codon optimised for yeasts. The reporter does not respond to chemicals which delay mitosis, and responds appropriately to the genetic regulation of DNA repair. Data is presented which demonstrate strain improvements appropriate to biosensor technology: improved signal to noise ratio, ease of data collection and uncomplicated material handling.