Is rapeseed really an allergenic plant? Popular myths versus scientific realities.
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Allergens↗
Biomedical subjects
D J Murphy
Publications and source records attributed to D J Murphy.
Production of novel oils in plants.
We have now isolated the great majority of genes encoding enzymes of storage oil biosynthesis in plants. In the past two years, particular progress has been made with acyltransferases, ketoacyl-acyl carrier protein synthetases and with desaturases and their relatives. In some cases, these enzymes have been reengineered to create novel products. Nevertheless, the single or multiple insertion of such transgenes into oil crops has not always led to the desired phenotype. We are only now beginning to appreciate some of the complexities of storage and membrane lipid formation, such as acyl group remodelling and the turnover of unusual fatty acids. This understanding will be vital for future attempts at the rational engineering of transgenic oil crops. In parallel with this, the domestication of plants already synthesising useful fatty acids should be considered as a real alternative to the transgenic approach to producing novel oil crops.
Fatty Acid Desaturases↗
Mechanisms of lipid-body formation.
Most organisms transport or store neutral lipids as lipid bodies - lipid droplets that usually are bounded by specific proteins and (phospho)lipid. Neutral-lipid bodies vary considerably in their morphology and are associated with an extremely diverse range of proteins. However, the mechanisms by which they are generated in plants, animals and microorganisms appear to share many common features: lipid bodies probably arise from microdomains of the endoplasmic reticulum (or the plasma membrane in prokaryotes) that contain lipid-biosynthesis enzymes, and their synthesis and size appear to be controlled by specific protein components.
Adipocytes↗
Temporal and spatial gene expression of cytochrome B5 during flower and fruit development in olives.
We report the characterisation of two cytochrome b5 genes and their spatial and temporal patterns of expression during development in olive, Olea europaea. A PCR-generated probe, based on a tobacco cytochrome b5 sequence, was used to isolate two full-length cDNA clones (cytochrome b5-15 and cytochrome b5-38) from a library derived from 13 WAF olive fruits. The cDNAs encoded proteins of 17.0 and 17.7 kDa, which contained all the characteristic motifs of cytochromes b5 from other organisms and exhibited 63% identity and 85% similarity with each other. The olive cytochrome b5-15 cDNA was then used as a probe for more detailed analysis. Southern blotting revealed a gene family of at least 4-6 members while northern blotting and in situ hybridisation showed a highly specific pattern of gene expression. Very low levels of cytochrome b5 mRNA were detected in tissues characterised by high rates of lipid accumulation, such as young expanding leaves, maturing seeds and ripening mesocarp. The cytochrome b5 genes were not induced at 6 degrees C and their response to ABA was relatively slow compared with fatty acid desaturase genes. In contrast, high levels of cytochrome b5 gene expression were found in young fruits at the pattern formation (globular/heart) stage of embryogenesis and in vascular and transmitting tissues of male and female reproductive organs. The data are consistent with a major role for cytochrome b5 in developmental processes related to plant reproduction in addition to being an electron donor to microsomal desaturases.
Abscisic Acid↗
Human SWI-SNF component BRG1 represses transcription of the c-fos gene.
Yeast and mammalian SWI-SNF complexes regulate transcription through active modification of chromatin structure. Human SW-13 adenocarcinoma cells lack BRG1 protein, a component of SWI-SNF that has a DNA-dependent ATPase activity essential for SWI-SNF function. Expression of BRG1 in SW-13 cells potentiated transcriptional activation by the glucocorticoid receptor, which is known to require SWI-SNF function. BRG1 also specifically repressed transcription from a transfected c-fos promoter and correspondingly blocked transcriptional activation of the endogenous c-fos gene. Mutation of lysine residue 798 in the DNA-dependent ATPase domain of BRG1 significantly reduced its ability to repress c-fos transcription. Repression by BRG1 required the cyclic AMP response element of the c-fos promoter but not nearby binding sites for Sp1, YY1, or TFII-I. Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb protein, transcriptional repression by BRG1 was weak unless wild-type Rb was also supplied. Interestingly, Rb-dependent repression by BRG1 was found to take place through a pathway that is independent of transcription factor E2F.
Activating Transcription Factors↗
Deaths related to intrapartum asphyxia. Denominators are needed before conclusions can be drawn.
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Asphyxia Neonatorum↗
Pain management and chemical dependency.
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Analgesics, Opioid↗
Branch retinal artery occlusion from a retained left atrial catheter 21 years after operation.
A case of branch retinal artery occlusion due to an embolus from a retained left atrial catheter is presented. Removal was accomplished by reoperation. Prompt removal of any retained intracardiac catheter is recommended.
Adult↗
Double-outlet right ventricle with complete atrioventricular canal.
A case of double-outlet right ventricle, unbalanced complete atrioventricular canal, hypoplastic aortic arch, coarctation of the aorta, and atrioventricular valve regurgitation was treated with a staged approach. At 4 days of life the patient underwent aortic arch repair, atrial septectomy, common atrioventricular valve regurgitation repair, and pulmonary artery banding. When she was 14 months of age a biventricular repair was accomplished by two-patch complete atrioventricular canal repair and arterial switch procedure.
Atrioventricular Node↗
A novel method for chronic measurement of pleural pressure in conscious rats.
Pleural pressures are used to evaluate lung function and are generally measured acutely in anesthetized animals. Previous attempts to measure pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal. These problems have been eliminated by developing a novel surgical procedure for placement of a pressure-sensitive catheter beneath the pleural surface. The catheter (attached to a radiotelemetry transmitter) is surgically implanted beneath the serosal layer of the esophagus within the thoracic cavity. This is accomplished by making a small incision in the serosal layer of the esophagus caudal to the diaphragm and advancing the catheter cranially into the thoracic cavity until pressure changes are maximal. The accuracy of these measurements was verified by comparison with direct pleural pressure measurements over the range of -3 to -34 cm H2O. The pleural pressure changes remained constant for at least 14 weeks following surgery, and there was no evidence of tissue damage or growth around the catheter. This novel method for measuring pleural pressure chronically in conscious rats will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated and simultaneous measurements of both ventilatory (breathing) patterns and lung function in conscious animals.
Animals↗
Biosynthesis, targeting and processing of oleosin-like proteins, which are major pollen coat components in Brassica napus.
The purpose of this study is to characterise the biosynthesis, targeting and processing of some of the major protein components of the pollen coat, or tryphine, of Brassica napus. The authors have N-terminally sequenced 11 of the most abundant pollen coat polypeptides, and nine of these sequences correspond to proteolytically cleaved products of seven oleosin-like genes, i.e. Oln B;1 to Oln B;6 and Oln B;11. The Oln B;11 gene product is co- or post-translationally targeted in vitro to canine microsomal membranes. This implies that the oleosin-like protein is targeted to the endoplasmic reticulum in tapetal cells in vivo. Affinity-purified antibodies raised against a 20-residue domain of Oln B;3 and B;4 gene products cross-reacted with full-length proteins of 45-48 kDa in early developing (< 2 mm to 5 mm) buds and anthers, but recognised truncated proteins of 32-38 kDa at later (4 mm to 7 mm) stages of development. The 45-48 kDa immunoreactive proteins were associated with a floating lipid body fraction obtained from a tapetal/locular fluid extract from maturing anthers and a major 48 kDa polypeptide from this fraction was confirmed by N-terminal sequencing to be a full length product of the Oln B;3 gene. Quantitative immunocytochemical studies showed that the full length 45-48 kDa oleosin-like proteins were specifically localised in the interior of tapetal cytoplasmic lipid bodies where they were associated with a regular hexagonal-like fibrous reticulum. No significant labelling of elaioplasts was observed. The same antibodies specifically labelled 32-38 kDa oleosin-like proteins on the extracellular pollen coat of maturing pollen grains. These results demonstrate for the first time that many of the major pollen coat proteins are derived from an endoproteolytic cleavage of precursor oleosin-like proteins that originally accumulate within the large cytoplasmic lipid bodies of tapetal cells.
Amino Acid Sequence↗
Neonatal risk factors for cerebral palsy in very preterm babies: case-control study.
OBJECTIVE: To identify neonatal risk factors for cerebral palsy among very preterm babies and in particular the associations independent of the coexistence of antenatal and intrapartum factors. DESIGN: Case-control study. SETTING: Oxford health region. SUBJECTS: Singleton babies born between 1984 and 1990 at less than 32 weeks' gestation who survived to discharge from hospital: 59 with cerebral palsy and 234 randomly selected controls without cerebral palsy. MAIN OUTCOME MEASURES: Adverse neonatal factors expressed as odds ratios and 95% confidence intervals. RESULTS: Factors associated with an increased risk of cerebral palsy after adjustment for gestational age and the presence of previously identified antenatal and intrapartum risk factors were patent ductus arteriosus (odds ratio 2.3; 95% confidence interval 1.2 to 4.5), hypotension (2.3; 1.3 to 4.7), blood transfusion (4.8; 2.5 to 9.3), prolonged ventilation (4.8; 2.5 to 9.0), pneumothorax (3.5; 1.6 to 7.6), sepsis (3.6; 1.8 to 7.4), hyponatraemia (7.9; 2.1 to 29.6) and total parenteral nutrition (5.5; 2.8 to 10.5). Seizures were associated with an increased risk of cerebral palsy (10.0; 4.1 to 24.7), as were parenchymal damage (32; 12.4 to 84.4) and appreciable ventricular dilatation (5.4; 3.0 to 9.8) detected by cerebral ultrasound. CONCLUSION: A reduction in the rate of cerebral palsy in very preterm babies requires an integrated approach to management throughout the antenatal, intrapartum, and neonatal periods.
Blood Transfusion↗
Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of beta-glucuronidase in transgenic Brassica plants.
To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5'-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene beta-glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5'-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.
Base Sequence↗
Intra- and extracellular lipid composition and associated gene expression patterns during pollen development in Brassica napus.
Pollen development in angiosperms is regulated by the interaction of products contributed by both the gametophytic (haploid) and sporophytic (diploid) genomes. In entomophilous species, lipids are major products of both sporophytic and gametophytic metabolism during pollen development. Mature pollen grains of Brassica napus are shown to contain three major acyl lipid pools as follows: (i) the extracellular tryphine mainly consisting of medium-chain neutral esters; (ii) the intracellular membranes, particularly endoplasmic reticulum, mainly containing phospholipids; and (iii) the intracellular storage lipids, which are mostly triacylglycerols. This paper reports on the kinetics of accumulation of these lipid classes during pollen maturation and the expression patterns of several lipid biosynthetic genes and their protein products that are differentially regulated in developing microspores/ pollen grains (gametophyte) and tapetal cells (sporophyte) of B. napus. Detailed analysis of three members of the stearoyl-ACP desaturase (sad) gene family by Northern blotting, in situ hybridization and RT-PCR showed that the same individual genes were expressed both in gametophytic and sporophytic tissues, although under different temporal regulation. In the tapetum, maximal expression of two marker genes for lipid biosynthesis (sad and ear) occurred at a bud length of 2-3 mm, and the corresponding gene products SAD and EAR were detected by Western blotting in 3-4 mm buds, coinciding with the maximal rates of tapetal lipid accumulation. These lipids are released following tapetal cell disintegration and are relocated to form the major structural component of the extracellular tryphine layer that coats the mature pollen grain. In contrast, in developing microspores/pollen grains, maximal expression of the lipid marker genes sad, ear, acp and cyb5 was at the 3-5 mm bud stages, with the SAD and EAR gene products detected in 4-7 mm buds. This pattern of expression coincided with accumulation of the intracellular storage and membrane lipid components of pollen. These results suggest that, although the same genes may be expressed in the sporophytic tapetal cells and in gametophytic tissues, they are regulated differentially leading to the production of the various contrasting lipidic structures that are assembled together to give rise to a viable, fertile pollen grain.
Brassica↗
Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds.
Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T2 seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10-1.14 g ml-1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/processing changes in the protein. Although oil-body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.
Brassica↗
An unusual complication of laparoscopic salpingostomy for ectopic pregnancy.
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Journal Article↗
Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting.
An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.
Amino Acid Sequence↗
Marginally effective medical care: ethical analysis of issues in cardiopulmonary resuscitation (CPR)
Outcomes from cardiopulmonary resuscitation (CPR) remain distressingly poor. Overuse of CPR is attributable to unrealistic expectations, unintended consequences of existing policies and failure to honour patient refusal of CPR. We analyzed the CPR outcomes literature using the bioethical principles of beneficence, non-maleficence, autonomy and justice and developed a proposal for selective use of CPR. Beneficence supports use of CPR when most effective. Non-maleficence argues against performing CPR when the outcomes are harmful or usage inappropriate. Additionally, policies which usurp good clinical judgment and moral responsibility, thereby contributing to inappropriate CPR usage, should be considered maleficent. Autonomy restricts CPR use when refused but cannot create a right to CPR. Justice requires that we define which medical interventions contribute sufficiently to health and happiness that they should be made universally available. This ordering is necessary whether one believes in the utilitarian standard or wishes medical care to be universally available on fairness grounds. Low-yield CPR fails justice criteria. Cardiopulmonary resuscitation should be performed when justified by the extensive outcomes literature; not performed when not desired by the patient or not indicated; and performed infrequently when relatively contraindicated.
Beneficence↗