Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase.

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.

Care home nursing: Co-creating curricular content with student nurses.

BACKGROUND: Meeting the complex care needs of an ageing population is a global issue and long term care settings, such as care homes, play an essential role. However, there is a crisis in the recruitment of registered nurses within care homes. Higher educational institutions have a critical part to play in addressing the crisis in recruitment in care homes and it is argued that student nurses can have a significant role to play in co-creating curricular content responsive to population need. OBJECTIVE: To co-create curricular content on care home nursing with student nurses. DESIGN: Co-creation through collaborative enquiry and a three stage thematic analysis. SETTING: Undergraduate, preregistration nursing programme in a university in the United Kingdom. PARTICIPANTS: Student nurses from Years One to Four undertaking a Bachelor in Nursing with Honours degree. METHODS: Six focus groups and two one to one interviews. RESULTS: Findings revealed predominantly negative attitudes towards care home nursing. Teaching and practice placements appeared to play a minor role in shaping students' attitudes but rather, gave the unspoken message that for the acquisition of necessary knowledge and skills, care homes were less important than other settings. Most students were initially averse to care home nursing as a career choice. During focus groups/interviews, views shifted from seeing care homes as places where you 'lose clinical skills' to places where there is 'a lot of responsibility', and also a potentially rewarding career choice. From this attitudinal shift, students made suggestions for developing better curricular content and more positive learning opportunities. CONCLUSIONS: A co-creative framework can create a space for mutual learning between students and staff about challenges and opportunities for equipping nurses to meet the needs of ageing populations. Student nurses are open to learning about care home nursing as part of their education and keen to have a more positive exposure.

Regulation of the C/EBP-related gene gadd153 by glucose deprivation.

gadd153 encodes a CCAAT/enhancer-binding protein (C/EBP)-related protein that lacks a functional DNA-binding domain. Since the gadd153 protein is capable of heterodimerizing with other C/EBPs, gadd153 may function as a negative regulator of these transcription factors. Here we examined the role of glucose in regulating gadd153 expression. We found that glucose deprivation markedly induces gadd153 mRNA levels in both HeLa and 3T3-L1 cells and that addition of D-(+)-glucose resulted in a rapid decrease of gadd153 mRNA. Similar induction and reversal of gadd153 expression were observed at the protein level. Because C/EBP alpha appears to play an important role in regulating genes involved in adipogenesis and energy metabolism, we examined gadd153 expression during the differentiation of 3T3-L1 preadipocytes and as a function of glucose utilization in differentiated adipocytes. Using a standard differentiation protocol that consisted of hormonal stimulation for 2 days followed by medium changes every 2 days thereafter, we observed that both C/EBP alpha and gadd153 mRNAs were elevated. However, C/EBP alpha induction occurred on day 3, while gadd153 expression was not seen until day 4, when the cells were fully differentiated. Frequent addition of fresh medium to the cells during the differentiation process, as well as supplementation of medium with glucose, reduced gadd153 expression without preventing C/EBP alpha expression or interfering with cellular differentiation. Thus, gadd153 expression is not essential for the process of adipocyte differentiation but is significantly influenced by the availability of glucose to the cell.

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.

Activation of the cholesterol pathway and Ras maturation in response to stress.

All cells depend on sterols and isoprenoids derived from mevalonate (MVA) for growth, differentiation, and maintenance of homeostatic functions. In plants, environmental insults like heat and sunlight trigger the synthesis of isoprene, also derived from MVA, and this phenomenon has been associated with enhanced tolerance to heat. Here, we show that in human prostate adenocarcinoma PC-3M cells heat shock leads to activation of the MVA pathway. This is characterized by a dose- and time-dependent elevation in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) activity, enhanced sterol and isoprenoid synthesis, and increased protein prenylation. Furthermore, prenylation and subsequent membrane localization of Ras, a central player in cell signaling, was rapidly induced following heat stress. These effects were dose-dependent, augmented with repeated insults, and were prevented by culturing cells in the presence of lovastatin, a competitive inhibitor of HMGR. Enhanced Ras maturation by heat stress was also associated with a heightened activation of extracellular signal-regulated kinase (ERK), a key mediator of both mitogenic and stress signaling pathways, in response to subsequent growth factor stimulation. Thus, activation of the MVA pathway may constitute an important adaptive host response to stress, and have significant implications to carcinogenesis.

Crystallization of the NADH-specific enoyl acyl carrier protein reductase from Brassica napus.

The tetrameric, NADH-dependent enoyl acyl carrier protein reductase from developing seeds of Brassica napus (oil seed rape) has been crystallized from solutions containing ammonium sulphate as the precipitant in the presence of NAD+ or NADH using the hanging drop method of vapour diffusion. The crystals belong to the tetragonal system and are in space group P4(2)2(1)2 with cell dimensions a = b = 70.5 A, c = 117.8 A. Considerations of the possible values of Vm indicate that the asymmetric unit contains a single subunit. The crystals are resistant to radiation damage and X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 1.9 A resolution. Determination of the structure of this enzyme will advance the understanding of the mechanisms of lipid biosynthesis in plants and provide an opportunity to study the interactions between this enzyme and its acyl carrier protein substrate.

Activation of heat shock transcription factor 1 in rat aorta in response to high blood pressure.

We have previously demonstrated that acute hypertension induces heat shock protein gene expression in rat arterial wall. Here we provide evidence that this induction is mediated through the activation of heat shock transcription factor 1 in response to high blood pressure. Rats subjected to restraint or immobilization stress displayed an acute elevation in systolic pressure accompanied by an increase in heat shock protein 70 mRNA expression. Consistent with the rapid time course of mRNA induction, an increase in binding activity to an oligonucleotide encompassing a consensus heat shock element sequence was seen in protein extracts from aorta of restrained rats as assessed with gel mobility shift assays. A similar increase in DNA binding activity was also observed in aortic extracts from rats treated with various hypertensive agents, including phenylephrine, angiotensin II, and vasopressin. That the DNA binding activity was attributed to heat shock factor 1 was shown through use of antibodies to the transcription factor that retarded the DNA-protein complexes in gel mobility supershift assays. Western blot analysis of heat shock factor 1 protein expression in aortic extracts showed a slower mobility form of the protein in hypertensive rats, indicative of an activated, presumably phosphorylated, form of the transcription factor. These findings support the view that heat shock factor 1 is responsible for induction of heat shock protein 70 in the arterial wall during acute hypertension, a response that is likely to play an important role in protecting arteries during hemodynamic stress.

Induction of mitogen-activated protein kinase phosphatase-1 during acute hypertension.

Recently, we demonstrated that elevated blood pressure activates mitogen-activated protein (MAP) kinases in rat aorta. Here we provide evidence that the vascular response to acute hypertension also includes induction of MAP kinase phosphatase-1 (MKP-1), which has been shown to function in the dephosphorylation and inactivation of MAP kinases. Restraint or immobilization stress, which leads to a rapid rise in blood pressure, resulted in a rapid and transient induction of MKP-1 mRNA followed by elevated MKP-1 protein expression in rat aorta. That the induction of MKP-1 by restraint was due to the rise in blood pressure was supported by the finding that several different hypertensive agents (phenylephrine, vasopressin, and angiotensin II) were likewise capable of eliciting the response, and sodium nitroprusside, a nonspecific vasodilator agent that prevented the acute rise in blood pressure in response to the hypertensive agents, abrogated MKP-1 mRNA induction. The in vivo effects could not be mimicked by treatment of cultured aortic smooth muscle cells with similar doses of the hypertensive agents. These findings support a role for MKP-1 in the in vivo regulation of MAP kinase activity during hemodynamic stress.

Fatty acid synthesis in developing leaves of Brassica napus in relation to leaf growth and changes in activity of 3-oxoacyl-ACP reductase.

In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3-oxoacyl-ACP reductase. Using a novel radio-chemical assay for 3-oxoacyl-ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post-emergence, but slowed after this point even though 3-oxoacyl-ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis.

Kinetic mechanism of NADH-enoyl-ACP reductase from Brassica napus.

Enoyl-ACP reductase, a component of fatty acid synthase, is a target for anti-microbial agents and herbicides. Here we demonstrate the kinetic mechanism to be a compulsory-order ternary complex with NADH binding before the acyl substrate. Matrix-assisted laser desorption ionisation mass spectrometry analysis of enzymatically and synthesised crotonyl-ACP substrate showed the former to contain a single acyl group, whereas the latter contained up to four additional crotonylations. The use of authentic crotonyl-ACP will be important in future kinetic and crystallographic studies.

Potentiation of heat stress-induced hsp70 expression in vivo by aspirin.

Studies in cultured cells have demonstrated that non-steroidal anti-inflammatory agents can potentiate heat-induced hsp70 expression through activation of HSF1 to a DNA binding state. We investigated the influence of aspirin on hsp70 expression in intact rats subjected to heat stress. Rats were injected intraperitoneally either with aspirin (100 mg/kg) or vehicle alone, 60 min prior to their placement at 37 degrees C or room temperature for 30 min. hsp70 mRNA expression was analyzed in lung, liver and kidney isolated from animals assigned to one of four different treatment paradigms; untreated controls, heat, aspirin, and aspirin-plus-heat. Comparison of hsp70 expression in the treatment groups revealed that in all tissues examined, aspirin-plus-heat treatment resulted in 3-4 fold higher levels of hsp70 mRNA relative to those seen with heat treatment alone. Little or no hsp70 mRNA expression was detected in the unheated groups, regardless of aspirin treatment. In keeping with the mRNA expression, Hsp70 protein levels were also elevated in aspirin-plus-heat treated animals. Aspirin treatment did not alter hsp70 protein expression in the absence of heat. In contrast to in vitro observations, aspirin treatment in vivo did not alter HSF1 DNA binding properties. Core body temperature measurements revealed that aspirin pretreatment enhanced the rise in body temperature seen in response to heat treatment. This increased hyperthermic response to heat stress probably accounts for the potentiation of hsp70 expression observed in aspirin-plus-heat treated rats. Given the widespread use of aspirin in humans within a dose range comparable to that used here, our findings are likely to have important physiological consequences.

Effects of perturbations of the hypothalamic-pituitary-adrenal axis on the acute phase response: altered C/EBP and acute phase response gene expression in lipopolysaccharide-treated rats.

In this study, we investigated the influence of long term perturbations of the hypothalamicpituitary-adrenal axis on the acute phase response elicited following lipopolysaccharide (LPS) challenge in rats. Specifically, we examined the effects of either long term absence of glucocorticoids (adrenalectomized rats treated with placebo chronic release pellets) or extended exposure to pharmacologic levels of glucocorticoids (adrenalectomized rats treated with dexamethasone chronic release pellets) on the expression of selected acute phase proteins and various members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. Both hypothalamic-pituitary-adrenal axis manipulations resulted in a reduction of the acute phase response as assessed by the LPS-mediated induction of acute phase proteins and C/EBP gene expression, with dexamethasone treatment exhibiting a greater inhibitory effect than adrenalectomy. Induction of hemopexin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, GADD153, C/EBP beta, and C/EBP delta mRNAs by LPS were all abolished in dexamethasone-treated rats. These findings have direct implications for patients undergoing chronic high dose glucocorticoid therapy.

Natural surfactant and hyperoxic lung injury in primates. I. Physiology and biochemistry.

Surfactant dysfunction contributes to the pathophysiology of adult respiratory distress syndrome (ARDS), and we hypothesized that surfactant treatment would improve experimental ARDS produced by continuous exposure to hyperoxia. Twelve healthy male baboons (10-15 kg) were anesthetized, paralyzed, and mechanically ventilated with 2.5 cmH2O positive end-expiratory pressure (PEEP) for 96 h. Baboons were divided into three groups: 1) the O2 group (n = 5) received 100% O2, 2) the surfactant group (n = 5) received 100% O2 and aerosolized porcine surfactant, and 3) a control group (n = 2) was ventilated at fractional concentration of inspired O2 of 0.21 for 96 h to control for effects of anesthesia and mechanical ventilation. Hemodynamic parameters were obtained every 12 h, and ventilation-perfusion (VA/Q) distribution was measured daily by multiple inert gas elimination technique. PEEP was increased once or twice daily to 10 cmH2O for 30 min to study its effects on measurements of VA/Q. At the end of experiments, lungs were obtained for biochemical analysis. Prolonged hyperoxia resulted in progressive worsening in VA/Q, hemodynamic deterioration, severe lung edema, and altered surfactant metabolism. Surfactant administration increased disaturated phosphatidylcholine in lavage fluid but did not improve lung edema or gas exchange. In the surfactant group, however, the addition of 10 cmH2O PEEP resulted in a greater degree of shunt reduction than did 2.5 cmH2O PEEP (47 vs. 31% in the O2 group, P < 0.05). We conclude that aerosolized porcine surfactant did not prevent pulmonary O2 injury in baboons, but it potentiated the shunt-reducing effect of PEEP.

Artificial surfactant attenuates hyperoxic lung injury in primates. I. Physiology and biochemistry.

Prolonged exposure to O2 causes diffuse alveolar damage and surfactant dysfunction that contribute to the pathophysiology of hyperoxic lung injury. We hypothesized that exogenous surfactant would improve lung function during O2 exposure in primates. Sixteen healthy male baboons (10-15 kg) were anesthetized and mechanically ventilated for 96 h. The animals received either 100% O2 (n = 6) or 100% O2 plus aerosolized artificial surfactant (Exosurf; n = 5). A third group of animals (n = 5) was ventilated with an inspired fraction of O2 of 0.21 to control for the effects of sedation and mechanical ventilation. Hemodynamic parameters were obtained every 12 h, and ventilation-perfusion distribution (VA/Q) was measured daily using a multiple inert-gas elimination technique. Positive end-expiratory pressure was kept at 2.5 cmH2O and was intermittently raised to 10 cmH2O for 30 min to obtain additional measurements of VA/Q. After the experiments, lungs were obtained for biochemical and histological assessment of injury. O2 exposures altered hemodynamics, progressively worsened VA/Q, altered lung phospholipid composition, and produced severe lung edema. Artificial surfactant therapy significantly increased disaturated phosphatidylcholine in lavage fluid and improved intrapulmonary shunt, arterial PO2, and lung edema. Surfactant also enhanced the shunt-reducing effect of positive end-expiratory pressure. We conclude that an aerosolized protein-free surfactant decreased the progression of pulmonary O2 toxicity in baboons.

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases of Escherichia coli C and Klebsiella pneumoniae M5a1 show very high N-terminal sequence homology.

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) dehydrogenase from Escherichia coli C and Klebsiella pneumoniae M5a1 have been purified and some of their properties studied. The apparent Km values for NAD and CHMS were 11.7 +/- 1.5 microM and 5.2 +/- 1.9 microM, respectively, for the K. pneumoniae enzyme, and 19.5 +/- 2.7 microM and 9.2 +/- 1.4 microM, respectively, for the E. coli enzyme. Both enzymes were optimally active at pH 7.5 in sodium phosphate buffer. They had subunit molecular weights of 52,000 (+/- 1000) and the native enzymes appeared to be dimers of identical subunits. The first 20 residues of their N-terminal amino acid sequences were 90% homologous. A degenerate oligonucleotide probe constructed to a six amino acid sequence common to both enzymes gave strong hybridization with DNA from E. coli strains B and W as well as with E. coli C and K. pneumoniae but little or no hybridization to DNA from E. coli K12 or Pseudomonas putida.

Some industrial developments and applications of multidimensional techniques.

Industrial analytical chemistry includes the measurement of the elemental composition and structure of molecules; the measurement of the concentration of specific molecules, atoms, and ions in contact with other molecules, atoms, and ions, the measurement of the energy and speed with which these reactions occur; and the separation of molecules, atoms, and ions specifically from other molecules, atoms and ions. It is also the measurement of the physical (interaction) and chemical (reaction) behavior of collections of molecules and how this behavior is controlled by the presence of other molecules and ions. Many excellent devices for separation and measurement have been developed to accomplish these tasks. Each of these attains a level of sensitivity and selectivity beyond which further improvement would be difficult. However, by coupling these techniques in various configurations, improved data can be generated in a short time span. Such techniques are often referred to as hyphenated, tandem, combined, or coupled. A more inclusive term is multidimensional techniques. In this paper, we briefly describe some of the most significant developments our laboratory has made in these and related techniques.

Improved free musculocutaneous flap survival with induction of heat shock protein.

The cellular response to a wide variety of stresses results in the synthesis of a family of stress response proteins termed heat shock proteins. Recent studies have demonstrated that heat shock proteins produced in response to an initial stress seem to protect against subsequent unrelated stresses. Importantly, hyperthermia-induced heat shock proteins provided protection from ischemia/reperfusion injury in several organ transplantation models. We hypothesized that free musculocutaneous flap survival could be improved by enhancing the flap's tolerance to relative ischemia by the prior induction of heat shock proteins. Accordingly, we determined the heat shock protein response in skin and muscle after systemic or local heating and examined the effect on free musculocutaneous flap survival in a rat model. Free musculocutaneous flaps incorporating thigh adductor muscles and a 2 x 6-cm2 skin paddle were transplanted to the ipsilateral groin in three groups of male Wistar rats. Systemically heated rats (n = 6) were anesthetized and incubated for 30 minutes at 42 degrees C 6 hours before free musculocutaneous tissue transfer. Locally heated rats (n = 6) were anesthetized, and their donor site anterior thigh was placed for 30 minutes on a heating block set at 44 degrees C 6 hours before free tissue transfer. Control rats (n = 5) did not have heating pretreatment but underwent identical anesthesia. Animals were sacrificed on postoperative day 3, at which time skin loss (cm2) and muscle viability, quantified by nitroblue tetrazolium staining time, were assessed in a blinded fashion. The skin and muscle from the free flap were analyzed for HSP72 mRNA and protein using quantitative Northern and Western blot techniques. All free musculocutaneous flaps were viable. However, the locally and systemically heated rats demonstrated a marked improvement of skin survival, which correlated with increased skin levels of HSP72. There were no differences in nitroblue tetrazolium muscle staining times or muscle levels of HSP72 among the three groups. These findings suggest that prior heat-induced heat shock proteins result in improvement in musculocutaneous flap survival, which may have direct clinical applications, especially in high-risk patients.

Effects of clenbuterol hydrochloride on pulmonary gas exchange and hemodynamics in anesthetized horses.

We evaluated the effects of clenbuterol HCl (0.8 micrograms/kg, of body weight, IV), a beta 2 agonist, on ventilation-perfusion matching and hemodynamic variables in anesthetized (by IV route), laterally recumbent horses. The multiple inert gas elimination technique was used to assess pulmonary gas exchange. Clenbuterol HCl induced a decrease in arterial oxygen tension (from 57.0 +/- 1.8 to 49.3 +/- 1.2 mm of Hg; mean +/- SEM) as a result of increased shunt fraction (from 6.6 +/- 2.1 to 14.4 +/- 3.1%) and ventilation to regions with high ventilation-perfusion ratios. In contrast, no changes in these variables were found in horses given sterile water. In horses given clenbuterol HCl, O2 consumption increased from 2.23 +/- 0.18 to 2.70 +/- 0.14 ml.min-1.kg-1, and respiratory exchange ratio decreased from 0.80 +/- 0.02 to 0.72 +/- 0.01. Respiratory exchange ratio and O2 consumption were not significantly modified in sterile water-treated (control) horses. Clenbuterol HCl administration was associated with increased cardiac index (from 57.4 +/- 4.0 to 84.2 +/- 6.3 ml.min-1.kg-1), decreased total peripheral vascular resistance (from 108.3 +/- 9.3 to 47.6 +/- 2.8 mm of Hg.s.kg.ml-1), and decreased pulmonary vascular resistance (from 31.3 +/- 3.8 to 13.6 +/- 0.7 mm of Hg.s.kg.ml-1). Our findings indicated that clenbuterol HCl may potentiate hypoxemia as a result of increased shunt fraction in horses anesthetized by the IV route, and caused changes in hemodynamic variables that were consistent with its ability to stimulate beta 2-adrenergic receptors.

Clinical credibility and the role of the nurse teacher.

This paper explores the role of the nurse teacher looking specifically at the issue of clinical credibility and the value, in terms of the student's optimal learning, of the nurse teacher teaching in both the classroom and the clinical setting. The concern for the nurse teacher to be clinically credible will be considered essentially in relation to how such a demand may be perceived by the nurse teacher. The various interpretations given to the need to be 'clinically credible' are considered and the implications of interpretations that do not entail actual direct care giving are discussed.

An evaluation of a clinical supervision programme.

In response to needs identified by community nurses in remote and isolated areas in North Argyll, Scotland, a programme of clinical supervision was implemented. The term 'practice support' was chosen by the community nurses for this project. The purpose was to provide peer support to counteract geographical isolation and to facilitate professional development through the use of reflective practice. Practice support was carried out over a period of 9 months. Pre-implementation and post-implementation questionnaires were used to evaluate the project.