A guide to deciphering the internal codes used by the tobacco industry.
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Documentation↗
Biomedical subjects
D Cullen
Publications and source records attributed to D Cullen.
Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer.
A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.
Biosensing Techniques↗
Observation of impurities in ice.
This paper presents a new method for determining the microstructural location of impurities in polycrystalline ice, which involves allowing the ice to sublimate under vacuum and then identifying the concentrated impurities using energy dispersive X-ray microanalysis in a low-vacuum scanning electron microscope. The method allows the presence of impurities in both the grain boundaries and the lattice of natural polycrystalline ice to be observed. Using this technique on natural ice, filaments consisting chiefly of NaCl were observed in some grain boundaries, small amounts of sulfur and chlorine were found in the grain interiors of the ice, and large concentrations of sulfur were observed in inclusions.
Electron Probe Microanalysis↗
Isolation and characterization of homokaryotic strains from the ligninolytic basidiomycete Ceriporiopsis subvermispora.
Genetic analyses of the lignin-degrading fungus Ceriporiopsis subvermispora is complicated by a dikaryotic nuclear condition and the absence of spore forms. Previous investigations had identified a family of closely related sequences encoding manganese peroxidase (MnP), but the relationship between genes and allelic variants could not be experimentally established. Addressing this issue, homokaryotic derivatives of C. subvermipora strain FP105752 were isolated from regenerated protoplasts. Designated CsA and CsB, their homokaryotic nature was established by polymerase chain reaction amplification and sequence analysis of the allelic variants of three MnP genes. Isoelectrofocusing revealed fewer MnP isoenzymes in filtrates of homokaryon cultures relative to the parental strain. The homokaryotic strains will simplify genetic analyses, particularly the identification of new genes.
Amino Acid Sequence↗
A PCR-based method for detection of potato pathogen, Helminthosporium solani, in silver scurf infected tuber tissue and soils.
Silver scurf caused by Helminthosporium solani causes significant economic losses in table stock, seed and processing potatoes. Specific polymerase chain reaction (PCR) primers, Hs1F1/Hs2R1, from H. solani were used for the amplification of a 447-bp product from 20 tissue samples and 54 single spore H. solani isolates, from eastern Canada (27 isolates), western Canada (13 isolates) and North Dakota in USA (14 isolates), but not from other potato fungal pathogens. In addition to PCR analysis, all 54 isolates were studied using conventional detection methods, visual disease symptoms and/or colony morphology and microscopic examination of the morphology of conidiophores and conidia. The PCR assay successfully detected H. solani and the PCR results correlated well with assessments based on conventional techniques. The detection of H. solani by PCR (1 day) is rapid and offers an alternative to the time consuming conventional diagnostic techniques (4-5 weeks). Nested PCR assay was necessary for the detection of H. solani in soils and thus can provide a sensitive technique to study the epidemiology of silver scurf in soils.
DNA Primers↗
Muscle glycogen supercompensation: absence of a gender-related difference.
Recently it has been reported that women do not have the capacity to accumulate supranormal levels of muscle glycogen when subjected to a carbohydrate (CHO) loading regimen [Tarnopolsky et al. (1995) J Appl Physiol 78:1360-1368]. Since, in this study, CHO intake relative to body mass in the female subjects was much lower than that in males, our primary aim was to re-examine this issue using subjects fed comparable amounts of CHO. Endurance-trained female and male subjects ingested 12 g CHO x kg(-1) lean body mass day(-1) in conjunction with the cessation of their daily physical training. A 3-day exposure to this diet resulted in a marked rise in muscle glycogen levels from [mean (SD)] 108 (15) mmol x kg(-1) wet weight to 193 (14) mmol x kg(-1) wet weight and 111 (16) m mol x kg(-1) wet weight to 202 (20) mmol x kg(-1) wet weight in the female participants during the post-menstrual and pre-menstrual phases of their menstrual cycle, respectively, and from 109 (27) mmol x kg(-1) wet weight to 183 (25) mmol x kg(-1) wet weight in males. We conclude that (1) female athletes have the capacity to accumulate supranormal levels of muscle glycogen, and (2) when exercise-trained males and females are fed comparable amounts of CHO relative to lean body mass, there is no gender-related difference in their ability to accumulate supranormal levels of muscle glycogen.
Adult↗
Mdm2 and the p53 pathway in human pituitary adenomas.
OBJECTIVE: Studies on pituitary tumours have failed to identify mutations in the tumour suppressor gene p53 suggesting that the protein identified is wild type. p21(WAF--1) is a downstream effector of p53 which promotes growth arrest. Mdm2 (mouse double minute) is a protein induced by wild type p53 and forms an autoregulatory feedback loop suppressing wild type p53 activity. The purpose of this study was to examine a group of pituitary tumours for expression of p53 and its two downstream effector proteins p21(WAF--1) and mdm2 and to compare this with their radiological invasive status and proliferative potential as assessed by Ki-67 expression. SUBJECTS AND METHODS: Sixty-nine tumours removed at transsphenoidal surgery were examined by immunocytochemistry using antibodies against p53, p21(WAF--1), mdm2 and Ki-67 (MIB-1). The invasive status of the tumours was determined from the preoperative CT/MRI scans. RESULTS: p53 was expressed in 42 of 69 (61%) pituitary adenomas but there was no relationship with either pituitary tumour invasive status (P = 0.71) or volume (P = 0.33). p53 expression correlated, however, with the proliferative state of the tumours as assessed by the MIB-1 labelling index (P = 0.0065). Invasive tumours had a higher growth fraction than non-invasive ones (P = 0.027). p21(WAF--1) was expressed in the nuclei of 58/69 (84%) pituitary adenomas and its expression correlated with that of p53 (r = 0.26, P = 0.03). Mdm2 was expressed in the cytoplasm of 46/69 (67%) tumours and this correlated with the nuclear staining for p53 (P = 0.022) while nuclear staining was seen in 32/69 (46%) tumours but this did not correlate significantly with nuclear p53 staining (P = 0.096). CONCLUSIONS: These results suggest that p53, p21(WAF--1) and mdm2 are all expressed in pituitary tumours suggesting that the p53 protein detected by immunocytochemistry is wild type. Expression of p53 is associated with tumours which have a higher proliferative status. The p53 activity is probably the result of upstream signals of local stresses mediated through either genetic change, cytokines, hypoxia or hormonal factors. Our results suggest, however, that the downstream pathway mediated through the activities of p21(WAF--1) and mdm2 may be dysfunctional in these tumours.
Adenoma↗
Isoenzyme multiplicity and characterization of recombinant manganese peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium.
We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) and Phanerochaete chrysosporium (H4) under control of the alpha-amylase promoter from Aspergillus oryzae in Aspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.
Aspergillus nidulans↗
A family's perception of a public hospital.
This article tells of one family's recent perceptions of major public hospital. Their story is a personal one hat revolves around the illness of a family member. Visiting their loved one every day meant they gained heightened awareness of the day-to-day running of the hospital. As the patient had to stay in hospital nger than originally anticipated the family members begin to observe details of contemporary hospital life not noticed on occasional visits. What they perceive leads them to question those often heard, and taken-for-granted, values of efficiency, accountability and competitiveness.
Aged↗
A homokaryotic derivative of a Phanerochaete chrysosporium strain and its use in genomic analysis of repetitive elements.
Analysis of complex gene families in the lignin-degrading basidiomycete Phanerochaete chrysosporium has been hampered by the dikaryotic nuclear condition. To facilitate genetic investigations in P. chrysosporium strain BKM-F-1767, we isolated a homokaryon from regenerated protoplasts. The nuclear condition was established by PCR amplification of five unlinked genes followed by probing with allele-specific oligonucleotides. Under standard nitrogen-limited culture conditions, lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities of the homokaryon were equivalent to those of the parental dikaryon. We used the homokaryon to determine the genomic organization and to assess transcriptional effects of a family of repetitive elements. Previous studies had identified an insertional mutation, Pce1, within lignin peroxidase allele lipI2. The element resembled nonautonomous class II transposons and was present in multiple copies in strain BKM-F-1767. In the present study, three additional copies of the Pce1-like element were cloned and sequenced. The distribution of elements was nonrandom; all localized to the same 3.7-Mb chromosome, as assessed by segregation analysis and Southern blot analysis of the homokaryon. Reverse transcription-PCR (RT-PCR) showed that Pce1 was not spliced from the lipI2 transcript in either the homokaryon or the parental dikaryon. However, both strains had equivalent lignin peroxidase activity, suggesting that some lip genes may be redundant.
Chromosome Mapping↗
Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis.
Glyoxal oxidase is a copper metalloenzyme produced by the wood-rot fungus Phanerochaete chrysosporium as an essential component of its extracellular lignin degradation pathways. Previous spectroscopic studies on glyoxal oxidase have demonstrated that it contains a free radical-coupled copper active site remarkably similar to that found in another fungal metalloenzyme, galactose oxidase. Alignment of primary structures has allowed four catalytic residues of glyoxal oxidase to be targeted for site-directed mutagenesis in the recombinant protein. Three glyoxal oxidase mutants have been heterologously expressed in both a filamentous fungus (Aspergillus nidulans) and in a methylotrophic yeast (Pichia pastoris), the latter expression system producing as much as 2 g of protein per liter of culture medium under conditions of high density methanol-induced fermentation. Biochemical and spectroscopic characterization of the mutant enzymes supports structural correlations between galactose oxidase and glyoxal oxidase, clearly identifying the catalytically important residues in glyoxal oxidase and demonstrating the functions of each of these residues.
Alcohol Oxidoreductases↗
Regulated expression of green fluorescent protein under the control of Aureobasidium pullulans xylanase gene xynA.
A mutant form of the jellyfish cDNA encoding green fluorescent protein (GFP) was fused to the promoter of the Aureobasidium pullulans xylanase gene xynA and the expression vector pxynEGFP was introduced into A. pullulans. In a manner consistent with regulation of the native xynA gene, gfp activity was induced by xylose and repressed by glucose. The marker may be useful for monitoring populations of A. pullulans in situ and for identifying transcriptional control elements of xynA.
Animals↗
Fluorescent labels, confocal microscopy, and quantitative image analysis in study of fungal biology.
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Actins↗
Organization and differential regulation of a cluster of lignin peroxidase genes of Phanerochaete chrysosporium.
The lignin peroxidases of Phanerochaete chrysosporium are encoded by a minimum of 10 closely related genes. Physical and genetic mapping of a cluster of eight lip genes revealed six genes occurring in pairs and transcriptionally convergent, suggesting that portions of the lip family arose by gene duplication events. The completed sequence of lipG and lipJ, together with previously published sequences, allowed phylogenetic and intron/exon classifications, indicating two main branches within the lip family. Competitive reverse transcription-PCR was used to assess lip transcript levels in both carbon- and nitrogen-limited media. Transcript patterns showed differential regulation of lip genes in response to medium composition. No apparent correlation was observed between genomic organization and transcript levels. Both constitutive and upregulated transcripts, structurally unrelated to peroxidases, were identified within the lip cluster.
Carbon↗
Simple subgingival impressions.
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Dental Impression Technique↗
Primary gastroduodenal prophylaxis with omeprazole for non-steroidal anti-inflammatory drug users.
AIM: To investigate the efficacy of omeprazole 20 mg o.m. as primary prophylaxis against non-steroidal anti-inflammatory drug (NSAID)-associated ulcer disease or dyspeptic symptoms. METHODS: A parallel group study compared patients randomized to receive omeprazole 20 mg o.m. or placebo as co-therapy with on-going NSAID treatment, over 6 months, in 19 specialist centres in Ireland, Hungary, France, the UK and the USA. One hundred and sixty-nine patients taking NSAIDs regularly, chronically and above defined minimum doses entered the trial. The main outcome measure was the development of gastric or duodenal ulcers detected endoscopically, the development of multiple erosions in the stomach or duodenum, or the onset of moderate or severe dyspeptic symptoms. RESULTS: The estimated probability of remaining free of these end-points for 6 months for patients taking omeprazole was 0.78 compared to 0.53 for placebo (P = 0.004). Fourteen patients receiving placebo (16.5%) developed 15 ulcers, comprising nine gastric and six duodenal ulcers, compared to three patients (3.6%) receiving omeprazole (all gastric ulcers). Logistic regression analysis showed that older patients were less likely, whilst those with rheumatoid arthritis were more likely, to remain free of NSAID-associated problems. CONCLUSIONS: Omeprazole is an effective agent for gastroduodenal prophylaxis in patients taking NSAIDs. Its main effect is to reduce the rate of development of gastric and duodenal ulcers.
Adolescent↗
Phanerochaete chrysosporium cellobiohydrolase and cellobiose dehydrogenase transcripts in wood.
The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium-colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation.
Base Sequence↗
Expression of phanerochaete chrysosporium genes encoding lignin peroxidases, manganese peroxidases, and glyoxal oxidase in wood
Expression of Phanerochaete chrysosporium genes encoding ligninolytic enzymes was assessed in wood. Poly(A) RNA was extracted from colonized wood chips by magnetic capture, and specific transcripts were quantified by competitive reverse transcriptase PCR. mRNA levels varied substantially among lignin peroxidase genes, and transcript patterns were dramatically different from those in previous studies with defined media.
Journal Article↗