Complex regulation of the TIR1/AFB family of auxin receptors.

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.

Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins.

The plant hormone auxin is central in many aspects of plant development. Previous studies have implicated the ubiquitin-ligase SCF(TIR1) and the AUX/IAA proteins in auxin response. Dominant mutations in several AUX/IAA genes confer pleiotropic auxin-related phenotypes, whereas recessive mutations affecting the function of SCF(TIR1) decrease auxin response. Here we show that SCF(TIR1) is required for AUX/IAA degradation. We demonstrate that SCF(TIR1) interacts with AXR2/IAA7 and AXR3/IAA17, and that domain II of these proteins is necessary and sufficient for this interaction. Further, auxin stimulates binding of SCF(TIR1) to the AUX/IAA proteins, and their degradation. Because domain II is conserved in nearly all AUX/IAA proteins in Arabidopsis, we propose that auxin promotes the degradation of this large family of transcriptional regulators, leading to diverse downstream effects.

The recent increase in Atlantic hurricane activity: causes and implications.

The years 1995 to 2000 experienced the highest level of North Atlantic hurricane activity in the reliable record. Compared with the generally low activity of the previous 24 years (1971 to 1994), the past 6 years have seen a doubling of overall activity for the whole basin, a 2.5-fold increase in major hurricanes (>/=50 meters per second), and a fivefold increase in hurricanes affecting the Caribbean. The greater activity results from simultaneous increases in North Atlantic sea-surface temperatures and decreases in vertical wind shear. Because these changes exhibit a multidecadal time scale, the present high level of hurricane activity is likely to persist for an additional approximately 10 to 40 years. The shift in climate calls for a reevaluation of preparedness and mitigation strategies.

Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response.

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, we show that plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.

Function of the ubiquitin-proteasome pathway in auxin response.

The plant hormone auxin regulates many aspects of growth and development. Despite the importance of this hormone, the molecular basis for auxin action has remained elusive. Recent advances using molecular genetics in Arabidopsis have begun to elucidate the mechanisms involved in auxin signaling. These results suggest that protein degradation by the ubiquitin pathway has a central role in auxin response.

Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana.

The plant hormone auxin regulates diverse aspects of plant growth and development. We report that in Arabidopsis, auxin response is dependent on a ubiquitin-ligase (E3) complex called SCFTIR1. The complex consists of proteins related to yeast Skp1p and Cdc53p called ASK and AtCUL1, respectively, as well as the F-box protein TIR1. Mutations in either ASK1 or TIR1 result in decreased auxin response. Further, overexpression of TIR1 promotes auxin response suggesting that SCFTIR1 is limiting for the response. These results provide new support for a model in which auxin action depends on the regulated proteolysis of repressor proteins.

A system for psychomotor evaluation; design, implementation and practice effects in volunteers.

This paper describes the design, implementation and assessment of PsychE, psychomotor evaluation system. Six standard tests are included: numeric vigilance, a dual task, probed memory recall, simple reaction time, choice reaction time and semantic long-term memory. The test presentations are described in detail. Practice effects were assessed in 10 healthy volunteers and were only evident in the performance measures for the simple reaction time test. For the remaining five tests, stable performance was reached within a single test session. The volunteers were healthy and most were regular users of computers. Therefore, the lack of practice effects cannot be assumed for the general population. A control group is essential for all studies using these tests. The system is implemented on an IBM-compatible personal computer and includes a database shell for the convenient collection, storage and analysis of performance data.

High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis.

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in Arabidopsis thaliana. When grown in the light at high temperature (29 degreesC), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20 degreesC. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.

The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast grr1p.

Genetic analysis in Arabidopsis has led to the identification of several genes that are required for auxin response. One of these genes, AXR1, encodes a protein related to yeast Aos1p, a protein that functions to activate the ubiquitin-related protein Smt3p. Here we report the identification of a new gene called TRANSPORT INHIBITOR RESPONSE 1 (TIR1). The tir1 mutants are deficient in a variety of auxin-regulated growth processes including hypocotyl elongation and lateral root formation. These results indicate that TIR1 is also required for normal response to auxin. Further, mutations in TIR1 display a synergistic interaction with mutations in AXR1, suggesting that the two genes function in overlapping pathways. The TIR1 protein contains a series of leucine-rich repeats and a recently identified motif called an F box. Sequence comparisons indicate that TIR1 is related to the yeast protein Grr1p and the human protein SKP2. Because Grr1p and other F-box proteins have been implicated in ubiquitin-mediated processes, we speculate that auxin response depends on the modification of a key regulatory protein(s) by ubiquitin or a ubiquitin-related protein.

Activated alleles of yeast SLN1 increase Mcm1-dependent reporter gene expression and diminish signaling through the Hog1 osmosensing pathway.

Two-component signal transduction systems involving histidine autophosphorylation and phosphotransfer to an aspartate residue on a receiver molecule have only recently been discovered in eukaryotes, although they are well studied in prokaryotes. The Sln1 protein of Saccharomyces cerevisiae is a two-component regulator involved in osmotolerance. Phosphorylation of Sln1p leads to inhibition of the Hog1 mitogen-activated protein kinase osmosensing pathway. We have discovered a second function of Sln1p by identifying recessive activated alleles (designated nrp2) that regulate the essential transcription factor Mcm1. nrp2 alleles cause a 5-fold increase in the activity of an Mcm1-dependent reporter, whereas deletion of SLN1 causes a 10-fold decrease in reporter activity and a corresponding decrease in expression of Mcm1-dependent genes. In addition to activating Mcm1p, nrp2 mutants exhibit reduced phosphorylation of Hog1p and increased osmosensitivity suggesting that nrp2 mutations shift the Sln1p equilibrium toward the phosphorylated state. Two nrp2 mutations map to conserved residues in the receiver domain (P1148S and P1196L) and correspond to residues implicated in bacterial receivers to control receiver phosphorylation state. Thus, it appears that increased Sln1p phosphorylation both stimulates Mcm1p activity and diminishes signaling through the Hog1 osmosensing pathway.

Flow dynamics through spinal needles.

We examined the flow pattern produced when liquid dye was actively injected into a fluid medium at various flows through five different commonly used spinal needles. At all flows, the Whitacre-type needles produced a directional stream exiting at an angle from the longitudinal axis. At intermediate rates the stream developed tracks which disappeared at faster rates. The Quincke needle always produced an undeviated stream of dye and did not form tracks at any flow rate. When a perspex plate (representing the spinal cord) was interposed in front of the needle, the dispersion of dye was always unidirectional from the Whitacre needle and bidirectional from the Quincke needle. The dye adhered to the surface of the plate as a concentrated film at slow rates and at faster rates it dispersed turbulently for both types of needle.

Isolation and analysis of the yeast TEA1 gene, which encodes a zinc cluster Ty enhancer-binding protein.

A genetic screen for mutants that affect the activity of internal regulatory sequences of Ty retrotransposons led to the identification of a new gene encoding a DNA-binding protein that interacts with the downstream enhancer-like region of Ty1 elements. The TEA1 (Ty enhancer activator) gene sequence predicts a protein of 86.9 kDa whose N terminus contains a zinc cluster and dimerization motif typical of the Gal4-type family of DNA-binding proteins. The C terminus encodes an acidic domain with a net negative charge of -10 and the ability to mediate transcriptional activation. Like other zinc cluster proteins, purified Tea1 was found to bind to a partially palindromic CGGNxCCG repeat motif located in the Ty1 enhancer region. The Ty1 Tea1 binding site has a spacing of 10 and is located near binding sites for the DNA-binding proteins Rap1 and Mcm1. Analysis of the phenotype of tea1 deletion mutants confirmed that the TEA1 gene is required for activation from the internal Ty1 enhancer characterized in this study and makes a modest contribution to normal Ty1 levels in the cell. Hence, Tea1, like Rap1, is a member of a small family of downstream activators in Saccharomyces cerevisiae. Further analysis of the Tea1 protein and its interactions may provide insight into the mechanism of downstream activation in yeast cells.

Skin conductance responses in patients sedated with midazolam or propofol.

We have measured the skin conductance response under resting conditions and to innocuous auditory stimuli in 45 patients receiving midazolam (group M), propofol (group P) or no sedative drug (group ND) before minor hand surgery under local anaesthesia. Administration of the sedative drugs was titrated to the end-point of slurring of speech and ptosis. The mean dose of midazolam was 0.06 (SD 0.01) mg kg-1 and the mean infusion rate of propofol was 2.2 (0.39) mg kg-1 h-1. Subjective ratings of anxiety and sedation were measured using visual analogue scales. These were similar in groups M and P and significantly different from those reported by group ND (P = 0.001-0.005). However, measures of skin conductance in group M were significantly lower than in group P (P = 0.002-0.013) and group ND (P = 0.004-0.016). These measures were similar in groups P and ND. Skin conductance measures were related significantly to anxiety scores only in groups M and ND (P < 0.05). We conclude that skin conductance is not a non-specific index of sedative-anxiolytic action and therefore is not useful in comparative studies of anxiolytic drugs that exert their effects by different pharmacological mechanisms.

Skin conductance responses to auditory stimuli and anticipatory responses before venepuncture in patients premedicated with diazepam or morphine.

We have measured the skin conductance response to innocuous auditory stimuli and the anticipatory response before venepuncture in 45 patients receiving diazepam, morphine or no premedication before general anaesthesia. Subjective ratings of anxiety and sedation were measured using visual analogue scales. Skin conductance was less in subjects receiving diazepam than in the other groups, and the pattern of change of skin conductance in this group indicated superior adaptation to the environment during presentation of the innocuous stimuli compared with the other groups. After warning of venepuncture there was a large increase in skin conductance in all groups. There was a significant relationship between anxiety and skin conductance in unpremedicated patients and those receiving diazepam.

Towards a standardized anaesthetic state using isoflurane and morphine.

In 34 patients undergoing major surgery, the inspired isoflurane concentration was adjusted by a control system designed to maintain systolic arterial pressure at a predetermined value. An empirical rule allowed additional morphine administration if the demand of the system for isoflurane was excessive. Satisfactory control of systolic arterial pressure was achieved in 31 patients and the anaesthetic state was clinically acceptable to an independent observer; no awareness was reported and the mean recovery time was 9.6 min. In these patients, control of systolic arterial pressure produced a pattern of clinical signs recognizable as general anaesthesia.

Role of Saccharomyces cerevisiae Rap1 protein in Ty1 and Ty1-mediated transcription.

Binding sites for the transcription factor Rap1 are widespread in the yeast genome. With respect to many, but not all, genes, Rap1p has an apparent activation function. Whether Rap1 is itself a transcriptional activator, or whether it is in some way required for activation by additional factors, is not clear. We have identified a previously unrecognized Rap1p binding site in the internal regulatory region of Ty1 elements. We demonstrate that this site is capable of binding Rap1 in vitro and that, in vivo, Rap1p plays an important regulatory role in Ty1 and Ty1-mediated adjacent gene expression. Our data suggest that in Ty1 elements, maximal levels of RAP1-mediated activation depend on the formation of a complex with Mcm1, an independent DNA-binding protein that functions in transcription as well as in DNA replication, and with a third factor, IBF, previously identified as a binding activity with a site situated between the Rap1p and Mcm1p binding sites in this region of Ty1 elements.

Oxford Optronix MPM 3S: a clinical assessment of a microvascular perfusion monitor.

The Oxford Optronix MPM 3S is a new microvascular perfusion monitor which is promoted as a device for use in the operating theatre. It uses a semiconductor laser diode and applies the Doppler principle to derive a semi-quantitative estimation of microvascular flow. We assessed this instrument with eight healthy volunteers who each performed eight different orthostatic arm manoeuvres while forearm skin blood flow was monitored. The different manoeuvres caused statistically significant changes in the instrument's reading which generally were consistent with expected changes in blood flow. The monitor also was assessed in the theatre environment with four anaesthetized patients. It proved easy to use, and was not subject to electrical interference from other equipment including short-wave diathermy. The major practical limitation of the technique is the semi-quantitative nature of the measurement. The instrument appears to have potential clinical uses in plastic and vascular surgery.

Towards a standardized anaesthetic state using enflurane and morphine.

General anaesthesia was maintained in 22 patients undergoing major surgery using a proportional-plus-integral control system. The system automatically adjusted the dose of enflurane according to the systolic arterial pressure (SAP). Additional morphine was administered if the system's demand for enflurane exceeded preset limits. In 21 patients, satisfactory control of SAP was achieved and anaesthesia was clinically adequate; no awareness was reported and the mean recovery time was 6.7 min (SD 3.5 min).

Nitrous oxide and formiminoglutamic acid: excretion in surgical patients and anaesthetists.

We have investigated the possible toxicity of nitrous oxide on vitamin B12 and its sequelae upon folic acid metabolism using the urine formiminoglutamic acid excretion test, an index of the functional state of folate metabolism. Ten control subjects not exposed to nitrous oxide and five patients receiving limb surgery under local anaesthesia excreted normal amounts of formiminoglutamic acid in urine for 6 days. Fifty patients received nitrous oxide anaesthesia for similar surgery and, of these, 22 had a dose-dependent increase in excretion on the first 2 days after operation. There were large individual variations. Exposure to 70% nitrous oxide appeared to cause abnormal metabolism of folate when exposure was greater than 90 min. Ten anaesthetists demonstrated normal excretion of formiminoglutamic acid; their exposure to nitrous oxide was typical of that in other studies of theatre environmental pollution.