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D J Murphy

Publications and source records attributed to D J Murphy.

At least 91 records · Page 5Linked to original sources
PubMed1994

Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene.

The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (Bn10) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter activity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P. Slocombe, I. Cummins, R.P. Jarvis, D.J. Murphy [1992] Plant Mol Biol 20: 151-155). Evidence is presented demonstrating the induction of desaturase mRNA by abscisic acid in the embryo.

Amino Acid Sequence↗
PubMed1994

Supportive family members of diabetic adults.

OBJECTIVE: Family members usually become involved during the course of care for a chronic illness. This study identified the diabetic adult's perceived supportive family member(s) and analyzed whether family participation was associated with the diabetic adults' level of metabolic control. METHODS: A telephone survey of 131 diabetic adults was conducted from a family practice residency office asking patients to identify family members participating in their diabetes care and to enumerate specific support activities. Demographic and metabolic control data were abstracted from patient records. RESULTS: Two broad categories of family participation exist. The first is the family health monitor (FHM), or internal "health expert" for the family. The other is the often distinct primary supportive family member; or "helper," defined as a family member who performs at least one supportive task in the care of the illness. Three-fourths of diabetic adults identified an FHM within their families. Eighty-seven percent of FHM's were women, usually adult daughters of diabetic women or wives of diabetic men. Forty-nine percent of diabetic women and 70% of diabetic men also identified a "helper." The most frequent helping tasks involved: dietary issues (48%), medication (23%), general support (15%) and blood sugar monitoring (9%). No relationship emerged between the presence or absence of an FHM or a helper and the level of metabolic control as measured by HbA1C level, which was categorized as "poor" in 55% of the sample. CONCLUSIONS: An FHM or some other helping family member is available to most diabetic adults in our patient population. The mere presence of an available FHM or other potential resource person is not necessarily related to a positive influence on metabolic control.

Adult↗
PubMed1994

GUIDe (Guidelines for the Use of Intensive Care in Denver): a community effort to define futile and inappropriate care.

Several issues force us to critically evaluate futile or inappropriate intensive care. These issues include cost control, quality of care, and professional and family integrity. The debate has progressed along three avenues: ethical discourse, prognostic scoring systems, and debate in the courts. Despite these arenas of discussion, a consensus about futile or inappropriate care has not been reached. The healthcare profession and the public need to work together to forge a consensus. We describe one model that facilitates this political process. Guidelines for the Use of Intensive Care in Denver (GUIDe) is a consortium of metropolitan Denver hospitals and other healthcare institutions whose goal is to develop guidelines for the use of futile or inappropriate intensive care. The building of consensus starts with subcommittees (adult intensive care, neonatal intensive care, and long-term care) that present proposals at plenary sessions. Other subcommittees (public liaison and legal subcommittees) facilitate dialogue with the public. Feedback from the plenary sessions, the greater medical community, and the public lead to proposal revisions. We expect to present hospitals with actual guidelines in approximately 3 yrs.

Colorado↗
PubMed1993

Expression and characterization of the N-terminal domain of an oleosin protein from sunflower.

Oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Although oleosins are amphipathic proteins, they are unlike bilayer membrane proteins since they are associated with a single lipid:water interface at the oil body surface. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain, flanked by relative polar C- and N-terminal domains. In the present study, we report the expression of the N-terminal domain of the 18-kDa oleosin isoform from sunflower as a recombinant fusion protein in Escherichia coli and the determination of its secondary structure using CD and Fourier transform infrared spectroscopy either as a purified but partially denatured peptide or reconstituted into liposomes. The structure derived from physical studies was then compared and assigned with those predicted from analysis of the primary sequence of the N-terminal domain. Based on data derived from CD spectroscopy analysis of purified and partially renatured N-terminal polypeptide, it contains about 10% alpha-helical structure, 20-30% beta-strand structure, approximately 8% beta-turn structure, and 60% random coil structure. However, analysis of the polypeptide reconstituted into liposomes showed an increased content of alpha-helical structure to about 20% and an increased beta-strand structure content to about 30-40%. Data derived from Fourier transform infrared spectroscopy studies and compared with the data predicted from the primary sequence showed the peptide is well structured with some antiparallel beta-strand structure from residues 2-9, parallel beta-strand structure from residues 30-37 and/or 42-49, and alpha-helical structure from residues 10-23 and/or 43-49. There is potential amphipathic alpha-helix from residues 10-23. Based on these results, the following model for the secondary structure of the N-terminal domain of sunflower oleosin can be proposed. Residues 2-9 would produce amphipathic antiparallel beta-strand structure. Residues 10-23 would produce an amphipathic alpha-helical structure. Residues 30-37 and/or 42-49 would give parallel beta-strand structure, or residues 42-49 could form a nonpolar alpha-helical structure that would insert into the oil matrix.

Amino Acid Sequence↗
PubMed1993

Differential, temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation.

The temporal and spatial expression of oleosin and delta 9-stearoyl-ACP desaturase genes and their products has been examined in developing embryos of rapeseed, Brassica napus L. var. Topas. Expression of oleosin and stearate desaturase genes was measured by in situ hybridisation at five different stages of development ranging from the torpedo stage to a mature-desiccating embryo. The temporal pattern of gene expression varied dramatically between the two classes of gene. Stearate desaturase gene expression was relatively high, even at the torpedo stage, whereas oleosin gene expression was barely detectable at this stage. By the stage of maximum embryo fresh weight, stearate desaturase gene expression had declined considerably while oleosin gene expression was at its height. In contrast to their differential temporal expression, the in situ labelling of both classes of embryo-specific gene showed similar, relatively uniform patterns of spatial expression throughout the embryo sections. Immunogold labelling of ultra-thin sections from radicle tissue with anti-oleosin antibodies showed similar patterns to sections from cotyledon tissue. However, whereas at least three oleosin isoforms were detectable on western blots of homogenates from cotyledons, only one isoform was found in radicles. This suggests that some of the oleosin isoforms may be expressed differentially in the various types of embryo tissue. The differential timing of stearate desaturase and oleosin gene expression was mirrored by similar differences in the timing of the accumulation of their ultimate products, i.e. storage oil and oleosin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Brassica↗
PubMed1993

Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.).

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.

Amino Acid Sequence↗
PubMed1993

Effects of adenosine agonists and antagonists on pulmonary ventilation in conscious rats.

1. The effects of intravenous injections of adenosine agonists and antagonists on pulmonary ventilation were investigated in conscious male Sprague-Dawley rats. 2. Adenosine agonists reduced pulmonary ventilation, whereas an adenosine antagonist increased pulmonary ventilation. 3. The adenosine-induced decrease in ventilation could be partially blocked by pretreatment with either an adenosine or opioid antagonist and completely blocked by a combined pretreatment with adenosine and opioid antagonists. 4. Thus, endogenous adenosine appears to have an inhibitory role in the control of pulmonary ventilation in conscious rats, and the mechanism of action appears to involve both adenosine and opioid receptors.

Adenosine↗
PubMed1993

A proposed classification code for farm and agricultural injuries.

A fundamental problem with classifying agriculturally related injury is that there is neither a rational nor a comprehensive scheme for grouping incidents into categories describing actual exposures encountered on farms and in agricultural work. Current surveillance systems are unable to differentiate between work that is related to farm production and work that is not, and to include all exposed persons in the surveillance. The proposed Farm and Agricultural Injury Classification Code is a step toward overcoming these problems. When it was applied to previously analyzed fatality data, 40% of cases previously identified as farm production work were reclassified into other categories.

Agricultural Workers' Diseases↗
PubMed1993

Characterization of a new class of oleosins suggests a male gametophyte-specific lipid storage pathway.

The expression and sequences of two related antherspecific cDNAs (I3 and C98) of Brassica napus (oilseed rape) were examined. These cDNAs were found to exhibit significant predicted amino acid sequence similarity with a group of seed-specific proteins, the oleosins, which are involved in oil body membrane structure. Pollen of B. napus also contains oil bodies, and the synthesis and accumulation of these organelles correlates with expression of the I3 and C98 transcripts. The protein content of purified pollen oil bodies was therefore examined in order to determine the presence or absence of possible oleosins. One major protein species of 14 kDa was identified and subjected to N-terminal amino acid sequence determination. The sequence of this pollen oil body associated protein is homologous to the predicted sequence of one of the anther-specific cDNs, I3, implying that this cDNA represents an anther-specific oleosin gene expressed in developing pollen. The expression of other genes active during seed oil body formation was also examined, and these were found to be specific to the sporophyte, inferring the existence of specific components of the pathway of lipid synthesis and storage in the male gametophyte. These possibilities and the relationship between different oleosin genes are discussed.

Amino Acid Sequence↗
PubMed1992

Secondary structures of a new class of lipid body proteins from oilseeds.

The three main isoforms of the 19-kDa lipid body proteins (oleosin) have been purified to homogeneity from embryos of rapeseed. The secondary structures of these proteins as derived from circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy were compared with the secondary structures predicted from the primary sequences. The salient feature of the primary sequence of all oleosins is its division into three defined structural domains: a central hydrophobic domain flanked on either side by relatively hydrophilic domains, respectively. Using a variety of predictive methods based on primary amino acid sequence data, the oleosins exhibited a high probability of beta-strand structure in the 70-residue central hydrophobic domain, with relatively little alpha-helical content. Secondary structure data derived from CD and FTIR were consistent with the predictions from primary sequence, showing that the oleosins contained about 45% beta-strand and 13% alpha-helical structure. Under high salt conditions, a 40-kDa polypeptide was obtained from purified preparations of the 19-kDa oleosins. The 40-kDa polypeptide has a very similar secondary structure, as analyzed by CD and FTIR, to that of the 19-kDa oleosins. This polypeptide is therefore probably a dimer of the 19-kDa oleosins that is formed in high salt environments. A model of the general structure of oleosins is proposed whereby the central hydrophobic domain of the protein with a predominantly beta-strand structure is embedded into the non-aqueous phase of lipid-bodies. This hydrophobic region is flanked by putative alpha-helical structures in the polar N- and C-terminal domains which are probably oriented at the lipid-water interface.

Amino Acid Sequence↗
PubMed1992

Cloning and characterisation of an oleosin gene from Brassica napus.

The sequence of an oleosin gene from Brassica napus has been determined. This gene contains a single intron of 437 bp and encodes a polypeptide of 195 amino acids. The oleosin gene product has an estimated molecular mass of 21.5 kDa and consists of a highly hydrophobic central domain flanked by relatively polar N- and C-terminal domains. The central domain is highly conserved between all oleosins sequenced to date and contains a run of periodically spaced leucine residues similar to that of a leucine-zipper motif. The gene has been shown to be expressed specifically in the embryo, maximally between 9 and 11 weeks after flowering, i.e. during the seed desiccation stage. Two transcriptional start sites have been mapped to -70 and -21 of the ATG and a putative ABA-responsive element and three repeated motifs have been identified in the promoter. These short promoter sequences could correspond to regulatory elements responsible for embryo-specific gene expression. Up to six genes exist in the oleosin gene family.

Amino Acid Sequence↗
PubMed1992

cDNA sequence of a sunflower oleosin and transcript tissue specificity.

Oleosins (oil body membrane proteins) of 20.5 and 18 kDa have been purified from sunflower (Helianthus annuus) seeds and polyclonal antibodies raised against them. The precipitated rabbit immunoglobulin fraction was purified by affinity chromatography on cyanogen bromide-activated Sepharose and specifically recognised polypeptides of 18 and 20.5 kDa in sunflower homogenate and oil body fractions assayed by western blotting. A near-full-length cDNA clone was isolated for the 20.5 kDa oleosin. The 694 bp cDNA contained an open reading frame of 534 bp, followed by an untranslated region of 81 bp and a poly(A) region of 70 bp. The open reading frame encoded a polypeptide of 19.8 kDa. Study of transcript localisation revealed message to be abundant in the embryo during the later stage of development and still present in the dry seed. No signal was observed in RNA prepared from expanding leaves.

Amino Acid Sequence↗