Dynamic behavior of Arabidopsis eIF4A-III, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia.

Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell.

Protein and energy metabolism in chronic bacterial infection: studies in melioidosis.

Chronic infection is often accompanied by a wasting process, the metabolic basis of which is not fully understood. The aims of the present study were to measure protein and energy metabolism in patients with melioidosis (a serious and antibiotic-refractory Gram-negative bacterial infection which is endemic in South-East Asia) in order to define the metabolic abnormalities that might contribute to wasting. Whole-body protein turnover was measured using the [(13)C]leucine technique, both in the fasted state and while consuming a high-energy meal. Resting energy expenditure was measured by indirect calorimetry, and total energy expenditure by the bicarbonate/urea method. Results were normalized for fat-free mass, as estimated from skinfold thickness. Protein turnover was increased in melioidosis patients compared with healthy controls during fasting (170.9 compared with 124.1 micromol x kg(-1) x h(-1); P=0.04), but the net rate of catabolism (22.2 compared with 20.5 micromol x kg(-1) x h(-1); P=0.77) and the anabolic response to feeding were similar in the two groups. Resting energy expenditure was higher in melioidosis patients compared with controls (191.4 and 157.3 kJ x kg(-1) x day(-1) respectively; P=0.04), but total energy expenditure (measured in a separate group of eight patients with melioidosis) was low (192.1 kJ x kg(-1) x day(-1)). In conclusion, this study found no evidence of metabolic causative factors, such as accelerated net protein catabolism during fasting, a blunted anabolic response to feeding or increased daily energy expenditure, and therefore suggests that reduced energy intake is the prime cause of wasting. The observed normal response to feeding should encourage nutritional approaches to prevent wasting.

ATP-dependent regulation of nuclear Ca(2+) levels in plant cells.

Localised alterations in cytoplasmic Ca(2+) levels are an integral part of the response of eukaryotic cells to a plethora of external stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca(2+) levels reflect the prevailing cytoplasmic Ca(2+) levels. Using nuclei prepared from carrot (Daucus carota L.) cells, we now show that Ca(2+) can be transported across nuclear membranes in an ATP-dependent manner and that over 95% of Ca(2+) is accumulated into a pool releasable by the Ca(2+) ionophore A.23187. ATP-dependent nuclear Ca(2+) uptake did not occur in the presence of ADP or ADPgammaS and was abolished by orthovanadate. Confocal microscopy of nuclei loaded with dextran-linked Indo-1 showed that the initial ATP-induced rise in [Ca(2+)] occurs in the nuclear periphery. The occurrence of ATP-dependent Ca(2+) uptake in plant nuclei suggests that alterations of intranuclear Ca(2+) levels may occur independently of cytoplasmic [Ca(2+)] changes.

Stimulation of myofibrillar protein degradation and expression of mRNA encoding the ubiquitin-proteasome system in C(2)C(12) myotubes by dexamethasone: effect of the proteasome inhibitor MG-132.

Addition of the synthetic glucocorticoid, dexamethasone (Dex) to serum-deprived C(2)C(12) myotubes elicited time- and concentration-dependent changes in N(tau)-methylhistidine (3-MH), a marker of myofibrillar protein degradation. Within 24 h, 100 nM Dex significantly decreased the cell content of 3-MH and increased release into the medium. Both of these responses had increased in magnitude by 48 h and then declined toward basal values by 72 h. The increase in the release of 3-MH closely paralleled its loss from the cell protein. Furthermore, Dex also decreased the 3-MH:total cell protein ratio, suggesting that myofibrillar proteins were being preferentially degraded. Incubation of myotubes with the peptide aldehyde, MG-132, an inhibitor of proteolysis by the (ATP)-ubiquitin (Ub)-dependent proteasome, prevented both the basal release of 3-MH (>95%) and the increased release of 3-MH into the medium in response to Dex (>95%). Northern hybridization studies demonstrated that Dex also elicited similar time- and concentration-dependent increases in the expression of mRNA encoding two components (14 kDa E(2) Ub-conjugating enzyme and Ub) of the ATP-Ub-dependent pathway. The data demonstrate that Dex stimulates preferential hydrolysis of myofibrillar proteins in C(2)C(12) myotubes and suggests that the ATP-Ub-dependent pathway is involved in this response.

Meloidogyne incognita Surface Antigen Epitopes in Infected Arabidopsis Roots.

Surface-coat epitopes of Meloidogyne incognita were detected in root tissues of Arabidopsis thaliana during migration and feeding site formation. A whole-mount root technique was used for immunolocalization of surface coat epitopes in A. thaliana, with the aid of a monoclonal antibody raised specifically against the outer surface of infective juveniles of M. incognita. The antibody, which was Meloidogyne-specific, recognized a fucosyl-bearing glycoprotein in the surface coat. During migration in host tissues the surface coat was shed, initially accumulating in the intercellular spaces next to the juvenile and later at cell junctions farther from the nematode. Upon induction of giant cell formation, the antibody bound to proximally located companion cells and sieve elements of the phloem.

Tissue protein synthesis rates in critically ill patients.

OBJECTIVES: The aims of this study were to simultaneously determine the in vivo rates of protein synthesis in skeletal muscle, peripheral blood lymphocytes, and serum albumin in critically ill patients; to establish whether a relationship between the responses of these tissues could be observed; and to demonstrate if a protein synthesis pattern characteristic of critical illness exists. DESIGN: Descriptive study. SETTING: Intensive care unit of a 1000-bed university hospital. PATIENTS: Fifteen patients treated in the intensive care unit. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Rates of tissue protein synthesis were determined in vivo once during the course of critical illness, using the flooding method with L-(2H5)phenylalanine. Protein synthesis in muscle was 1.49 +/- 0.16%/day; in circulating lymphocytes (i.e., mononuclear cells), protein synthesis was 11.10 +/- 1.82%/day. Albumin synthesis was 12.81 +/- 1.23%/day when expressed as the fractional rate, and was 184 +/- 19 mg/kg/day when expressed as the absolute rate. CONCLUSIONS: The individual tissues responded differently to trauma, and showed a wide range of values. The responses were not significantly correlated with each other and no pattern of tissue protein synthesis characteristic of critical illness was observed. However, both muscle protein and albumin synthesis rates correlated with metabolic status and clinical indices of the severity of illness.

Ca2+ oscillations in plant cells: initiation by rapid elevation in cytosolic free Ca2+ levels.

Temporal increases in intracellular [Ca2+] are now recognized to be key triggers for a wide range of important physiological events in eukaryotic cells. In mammalian cells, signal-induced Ca2+-elevations have been found to be of a pulsatile nature and Ca2+ spikes display a high degree of spatiotemporal complexity. In plant cells a similar picture is beginning to emerge. To investigate the occurrence of pulsatile Ca2+ signals in plant cells we studied alterations of [Ca2+] in the tip region of pollen tubes from poppy (Papaver rhoeas). Time-Resolved Laser Scanning Confocal Microscopy of pollen tubes microinjected with the Dextran-linked Ca2+-indicator dyes Calcium Green or Indo-1 revealed that highly regular Ca2+ oscillations occur in these cells. We further demonstrate that artificial elevation of cytosolic Ca2+ by photolysis of caged-Ca2+ (Nitr-5) can trigger the onset of oscillations.

Effect of a short-term infusion of glutamine on muscle protein metabolism postoperatively.

The acute effect of a short-term postoperative infusion of glucose supplemented with glutamine (0.285 g/kg body weight), on muscle protein metabolism, was studied by analyses of free amino acid concentrations and determinations of protein synthesis. A glutamine-glucose infusion was given for 5.5 h to 6 patients 2-3 days after elective surgery for colon cancer. The free glutamine concentration was 5.72 +/- 0.96 mmol/kg wet weight (ww) before and 6.14 +/- 1.10 mmol/kg ww 4 h after the glutamine infusion. The rate of protein synthesis was 1.26 +/- 0.15%/24 h before the infusion and 1.12 +/- 0.16%/24 h during its latter part. The percentage of polyribosomes was 42.2 +/- 3.4% before and 40.9 +/- 1.3% after the infusion. The results showed no difference in these biochemical parameters, indicating that a short-term infusion of glutamine given postoperatively is insufficient to affect protein metabolism in human skeletal muscle.

N tau-methylhistidine turnover in skeletal muscle cells measured by GC-MS.

A method that employs gas chromatography-mass spectrometry has been developed to measure N tau-methylhistidine (3-methylhistidine; 3-MH) synthesis and release from skeletal muscle myotubes in vitro. It shows excellent linearity (0.9999) over the range studied (0-4 nmol), high recovery (92.6%), and low coefficient of variation (1.6%). 3-MH release from myotubes was essentially linear over a 96-h incubation, whereas the loss of 3-MH from cell protein accelerated with increasing time, an effect due, at lest in part, to decreasing rates of total protein synthesis. When incubated in either glutamine-free or methionine-free medium for 48 h, 3-MH in cell protein and appearing in the medium were greatly reduced compared with the 48-h controls, suggesting that hypertrophy was greatly reduced. Similar but lesser trends were observed with adenosine 3',5' -cyclic monophosphate. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) appeared to both stimulate 3-MII synthesis and inhibit its release during a 48-h incubation. The development of this method facilitates detailed investigation into the mechanisms through which agents such as TPA regulate myofibrillar protein degradation.

Police referrals to a psychiatric hospital: indicators for referral and psychiatric outcome.

Studies of police psychiatric referrals in the USA and the UK generally show these patients to be ill and in need of care. There are, however, no published Australian studies and such findings may not be validly generalised. This prospective study of consecutive police psychiatric referrals in Adelaide reports psychiatric assessment in 92 cases and observations by police in 69 of these, with no evidence of selection bias. The most common reason for referral was threat of self harm (28%). Mental illness was deemed to be present in 49% and the most common clinical description was "situational crisis" (29%). Schizophrenia was diagnosed in 18%. Clinicians viewed 19% of referrals as inappropriate. Increased relative odds for mental illness were associated with police accounts of psychotic symptoms, and decreased odds with threat of self harm and violence. Increased odds for admission were associated with language difficulties and damage to own property, decreased odds with threat to others, threat of suicide, and threat to self injury. There were 14 cases where possible charges were not being pursued: of these 7 were regarded as ill and 4 were regarded as inappropriate referrals. The rates of major disorders are lower than in other published work. It is proposed that this can be explained by relative ease of referral by police to psychiatry and flexible acceptance criteria.

The effect of acute and chronic administration of the beta-agonist, cimaterol, on protein synthesis in ovine skin and muscle.

The action of intravenous infusion of the beta-agonist cimaterol (2.5 mg/d) on whole-body N retention and protein synthesis in peripheral tissues was examined in growing sheep. Wool growth was determined from skin patch clippings and adjusted to total fibre production. Protein synthesis was measured, using sequential large dose injections of [1-13C]valine, leucine and phenylalanine and then [ring-d5]phenylalanine, on biopsy samples from skin and m. longissimus dorsi taken before beta-agonist administration, at day 3 and day 15 of cimaterol infusion, and 15 d after withdrawal of the drug. Cimaterol increased total N retention by 1.9-2.3 g N/d (P < 0.01) over three successive 5 d periods. In contrast, wool growth was significantly reduced by 0.7 g N/d (P < 0.001) and the proportion of total N retained in wool declined from 0.71 to 0.25 (P < 0.01). The reduction in wool growth was accompanied by a decrease in protein fractional synthesis rate (FSR) in skin (11.6 v. 6.3%/d, P < 0.01). Muscle protein FSR, on the other hand, was markedly stimulated during cimaterol infusion (1.45 v. 3.01%/d, P < 0.001) as was RNA concentration (P < 0.001), RNA:protein (P < 0.001) and protein:DNA (P < 0.05). The estimated increase in total protein synthesis in muscle (+24 to 30 g/d) due to cimaterol administration was counterbalanced by reductions for skin (-25 to 27 g/d); this may account for the lack of changes in whole-body protein synthesis following beta-agonist administration reported in other studies. Although N retention rapidly returned to control values following withdrawal of the drug, both wool growth and skin protein synthesis remained depressed, while muscle protein FSR declined, but not to pre-treatment values. These results suggest a persistent action of cimaterol, but whether this is a function of residue concentrations or long-term metabolic responses is not known.

Glycerol exchange across the human forearm assessed by a combination of tracer and arteriovenous exchange techniques.

1. Whole-body kinetics and regional exchange of glycerol across forearm muscle were assessed in eight lean subjects by a combination of a tracer method (infusion of [2H5]glycerol) and arteriovenous catheterization. 2. During an apparent steady state, the enrichment of glycerol in deep venous blood from the muscle bed of the forearm was about half (4.40 +/- 1.72 atom per cent excess) that observed in arterialized blood (8.41 +/- 4.30 atom per cent excess). Under the same conditions, the circulating concentrations of glycerol in arterialized (91 +/- 24 mumol/l) and venous (87 +/- 32 mumol/l) blood were similar. 3. In a further group of 37 subjects it was found that about half had a positive arteriovenous concentration difference and the other had half a negative arteriovenous concentration difference (mean -1.6 +/- 11.9 mumol/l; range -25 to +22 mumol/l). 4. These results suggest: (a) that human muscle does not always release glycerol and may take it up; (b) that there is substantial isotopic exchange of glycerol across forearm muscle tissue, which is not reflected by the net exchange of glycerol; this could be due to slow equilibrium of enriched glycerol from the circulation, with unenriched free glycerol in the muscle pool, or due to the simultaneous metabolic utilization of enriched glycerol and metabolic production of unenriched glycerol; (c) that the estimation of glycerol flux rates is strongly dependent on whether the blood is arterialized or deep venous.

The effects of temperature on contractile mechanisms of rainbow trout (Salmo gairdneri) intestine.

Experiments were designed to determine whether contractility of trout smooth muscle in vitro varied with temperature and if changes occurred at the receptor or intracellular levels. The role of calcium in contractility at various temperatures was also investigated. Isolated trout intestinal segments, approximately 2 cm in length, were suspended isometrically under 2 g tension in 10-mL organ baths containing trout Ringer's solution aerated with O2 and CO2 (95:5). Contractions of trout intestine were not statistically different at 10 and 20 degrees C for carbachol, 5-hydroxytryptamine, and KCl. However, the efficacy, but not the potency, of each agonist was decreased at 2 degrees C. Receptor-induced contractions were reduced to a greater extent at 2 degrees C and did not recover to the same extent when returned to 10 degrees C in comparison with those induced by depolarization. The calcium source for contractility was also dependent on temperature. As temperatures increased, utilization of intracellular calcium increased, as indicated by increased contractility in the absence of extracellular calcium. Thus, low temperatures decrease smooth muscle contractility by affecting receptor-mediated events rather than the intracellular contractile mechanisms. Receptor-operated agonists appear to have a higher capability of using intracellular calcium than depolarizing agents.

Measurement of the rate of protein synthesis in muscle of postabsorptive young men by injection of a 'flooding dose' of [1-13C]leucine.

1. The 'flooding dose' technique for measuring the rate of protein synthesis in tissues in vivo involves the injection of a large amount of unlabelled amino acid together with the tracer to minimize differences in isotopic enrichment of the free amino acid in plasma and tissue compartments. This approach has been investigated in human muscle by taking biopsies from postabsorptive male volunteers given [1-13C]leucine. 2. Intravenous injection of 4 g of unlabelled leucine resulted in a rapid rise in free leucine concentration of seven- to eleven-fold in plasma and five-fold in muscle. Values were still elevated by two-fold after 2 h. 3. Five minutes after injection of [1-13C]leucine (0.05 g/kg) the isotopic enrichment of plasma leucine was 82% that of the injected material, falling to 44% at 120 min. The enrichment of free leucine in sequential muscle biopsies was close to that in plasma and almost identical to that for plasma alpha-ketoisocaproate. 4. The rate of protein synthesis was determined from the increase in leucine enrichment in protein of muscle biopsies taken before and 90 min after injection of [1-13C]leucine (0.05 g/kg; 19 or 39 atom% excess) and the average plasma alpha-ketoisocaproate enrichment over this period (taken to represent muscle free leucine). The mean rate of muscle protein synthesis in 10 subjects was 1.95 (SEM 0.12) %/day. Rates of protein synthesis calculated from plasma leucine as precursor enrichment were only 5% lower than those calculated from plasma alpha-ketoisocaproate.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug information network in the United Kingdom National Health Service.

The drug information network is the United Kingdom National Health Service (NHS) is described. The NHS was reorganized in 1974 resulting in the creation of 14 regions, which were further divided into areas. Drug information services provided within the regions and coordination between regions have evolved into a decentralized national drug information network. In this network, regional centers provide support to their area centers; difficult questions received by area centers may be referred to the regional center. Additionally, some regional centers have become specialized in particular fields. All regional centers have access to data held by other regional centers. Activity is further coordinated by a literature abstracting service in which each regional center has certain responsibilities; a code of practice for drug information pharmacists; production of specialty bulletins; and standardized education and training. Future plans for expanded services are described.