Neutrophil gelatinase-associated lipocalin: its response to hypoxia and association with acute mountain sickness.

Acute Mountain Sickness (AMS) is a common clinical challenge at high altitude (HA). A point-of-care biochemical marker for AMS could have widespread utility. Neutrophil gelatinase-associated lipocalin (NGAL) rises in response to renal injury, inflammation and oxidative stress. We investigated whether NGAL rises with HA and if this rise was related to AMS, hypoxia or exercise. NGAL was assayed in a cohort (n = 22) undertaking 6 hours exercise at near sea-level (SL); a cohort (n = 14) during 3 hours of normobaric hypoxia (FiO2 11.6%) and on two trekking expeditions (n = 52) to over 5000 m. NGAL did not change with exercise at SL or following normobaric hypoxia. During the trekking expeditions NGAL levels (ng/ml, mean ± sd, range) rose significantly (P < 0.001) from 68 ± 14 (60-102) at 1300 m to 183 ± 107 (65-519); 143 ± 66 (60-315) and 150 ± 71 (60-357) at 3400 m, 4270 m and 5150 m respectively. At 5150 m there was a significant difference in NGAL between those with severe AMS (n = 7), mild AMS (n = 16) or no AMS (n = 23): 201 ± 34 versus 171 ± 19 versus 124 ± 12 respectively (P = 0.009 for severe versus no AMS; P = 0.026 for mild versus no AMS). In summary, NGAL rises in response to prolonged hypobaric hypoxia and demonstrates a relationship to the presence and severity of AMS.

Early increased expression of a cyclin-dependant protein kinase (LtCDKA1;1) during inflorescence initiation of the long day grass Lolium temulentum.

Knowing where and when different genes express at the shoot apex during the transition to flowering will help in understanding this developmental switch. The CDKA family of serine/threonine kinase genes are appropriate candidates for such developmental switching as they are involved in the regulation of the G1/S and G2/M boundaries of the cell cycle (see review by Dudits et al. 2007) and so could regulate increases of cell division associated with flowering. Furthermore, in rice stems the gibberellin (GA) class of plant growth regulators rapidly upregulate CDKA expression and cell division. Thus, CDKA expression might be linked to the florigenic action of GA as a photoperiodically-generated, signal. For the grass Lolium temulentum L., we have isolated an LtCDKA1;1 gene, which is upregulated in shoot apices collected soon after the start of a single florally inductive long day (LD). In contrast to weak expression of LtCDKA1;1 in the vegetative shoot apex, in situ and PCR-based mRNA assays and immunological studies of its protein show very rapid increases in the apical dome at the time that florigenic signals arrive at the apex (<6h after the end of the LD). By ~54h LtCDKA1;1 mRNA is localised to the floral target cells, the spikelet primordia. Later both LtCDKA1;1 mRNA and protein are most evident in floret meristems. Only ~10% of cells within the apical dome are dividing at any time but the LD increase in LtCDKA1;1 may reflect an early transient increase in the mitotic index (Jacqmard et al. 1993) as well as a later increase when spikelet primordia form. Increased expression of an AP1-like gene (LtMADS2) follows that of LtCDKA1;1. Overall, LtCDKA1;1 is a useful marker of both early florigenic signalling and of later morphological/developmental aspects of the floral transition.

Inhibition of tiller bud outgrowth in the tin mutant of wheat is associated with precocious internode development.

Tillering (branching) is a major yield component and, therefore, a target for improving the yield of crops. However, tillering is regulated by complex interactions of endogenous and environmental signals, and the knowledge required to achieve optimal tiller number through genetic and agronomic means is still lacking. Regulatory mechanisms may be revealed through physiological and molecular characterization of naturally occurring and induced tillering mutants in the major crops. Here we characterize a reduced tillering (tin, for tiller inhibition) mutant of wheat (Triticum aestivum). The reduced tillering in tin is due to early cessation of tiller bud outgrowth during the transition of the shoot apex from the vegetative to the reproductive stage. There was no observed difference in the development of the main stem shoot apex between tin and the wild type. However, tin initiated internode development earlier and, unlike the wild type, the basal internodes in tin were solid rather than hollow. We hypothesize that tin represents a novel type of reduced tillering mutant associated with precocious internode elongation that diverts sucrose (Suc) away from developing tillers. Consistent with this hypothesis, we have observed upregulation of a gene induced by Suc starvation, downregulation of a Suc-inducible gene, and a reduced Suc content in dormant tin buds. The increased expression of the wheat Dormancy-associated (DRM1-like) and Teosinte Branched1 (TB1-like) genes and the reduced expression of cell cycle genes also indicate bud dormancy in tin. These results highlight the significance of Suc in shoot branching and the possibility of optimizing tillering by manipulating the timing of internode elongation.

The nature of floral signals in Arabidopsis. I. Photosynthesis and a far-red photoresponse independently regulate flowering by increasing expression of FLOWERING LOCUS T (FT).

Arabidopsis flowers in long day (LD) in response to signals transported from the photoinduced leaf to the shoot apex. These LD signals may include protein of the gene FLOWERING LOCUS T (FT) while in short day (SD) with its slower flowering, signalling may involve sucrose and gibberellin. Here, it is shown that after 5 weeks growth in SD, a single LD up-regulated leaf blade expression of FT and CONSTANS (CO) within 4-8 h, and flowers were visible within 2-3 weeks. Plants kept in SDs were still vegetative 7 weeks later. This LD response was blocked in ft-1 and a co mutant. Exposure to different LD light intensities and spectral qualities showed that two LD photoresponses are important for up-regulation of FT and for flowering. Phytochrome is effective at a low intensity from far-red (FR)-rich incandescent lamps. Independently, photosynthesis is active in an LD at a high intensity from red (R)-rich fluorescent lamps. The photosynthetic role of a single high light LD is demonstrated here by the blocking of the flowering and FT increase on removal of atmospheric CO(2) or by decreasing the LD light intensity by 10-fold. These conditions also reduced leaf blade sucrose content and photosynthetic gene expression. An SD light integral matching that in a single LD was not effective for flowering, although there was reasonable FT-independent flowering after 12 SD at high light. While a single photosynthetic LD strongly amplified FT expression, the ability to respond to the LD required an additional but unidentified photoresponse. The implications of these findings for studies with mutants and for flowering in natural conditions are discussed.

The nature of floral signals in Arabidopsis. II. Roles for FLOWERING LOCUS T (FT) and gibberellin.

Signals produced in leaves are transported to the shoot apex where they cause flowering. Protein of the gene FLOWERING LOCUS T (FT) is probably a long day (LD) signal in Arabidopsis. In the companion paper, rapid LD increases in FT expression associated with flowering driven photosynthetically in red light were documented. In a far red (FR)-rich LD, along with FT there was a potential role for gibberellin (GA). Here, with the GA biosynthesis dwarf mutant ga1-3, GA(4)-treated plants flowered after 26 d in short days (SD) but untreated plants were still vegetative after 6 months. Not only was FT expression low in SD but applied GA bypassed some of the block to flowering in ft-1. On transfer to LD, ga1-3 only flowered when treated simultaneously with GA, and FT expression increased rapidly (<19.5 h) and dramatically (15-fold). In contrast, in the wild type in LD there was little requirement for GA for FT increase and flowering so its endogenous GA content was near to saturating. Despite this permissive role for endogenous GA in Columbia, RNA interference (RNAi) silencing of the GA biosynthesis gene, GA 20-OXIDASE2, revealed an additional, direct role for GA in LD. Flowering took twice as long after silencing the LD-regulated gene, GA 20-OXIDASE2. Such independent LD input by FT and GA reflects their non-sympatric expression (FT in the leaf blade and GA 20-OXIDASE2 in the petiole). Overall, FT acts as the main LD floral signal in Columbia and GA acts on flowering both via and independently of FT.

Selective deactivation of gibberellins below the shoot apex is critical to flowering but not to stem elongation of Lolium.

Gibberellins (GAs) cause dramatic increases in plant height and a genetic block in the synthesis of GA(1) explains the dwarfing of Mendel's pea. For flowering, it is GA(5) which is important in the long-day (LD) responsive grass, Lolium. As we show here, GA(1) and GA(4) are restricted in their effectiveness for flowering because they are deactivated by C-2 hydroxylation below the shoot apex. In contrast, GA(5) is effective because of its structural protection at C-2. Excised vegetative shoot tips rapidly degrade [14C]GA(1), [14C]GA(4), and [14C]GA(20) (>80% in 6 h), but not [14C]GA(5). Coincidentally, genes encoding two 2beta-oxidases and a putative 16-17-epoxidase were most expressed just below the shoot apex (<3 mm). Further down the immature stem (>4 mm), expression of these GA deactivation genes is reduced, so allowing GA(1) and GA(4) to promote sub-apical stem elongation. Subsequently, GA degradation declines in florally induced shoot tips and these GAs can become active for floral development. Structural changes which stabilize GA(4) confirm the link between florigenicity and restricted GA 2beta-hydroxylation (e.g. 2alpha-hydroxylation and C-2 di-methylation). Additionally, a 2-oxidase inhibitor (Trinexapac Ethyl) enhanced the activity of applied GA(4), as did limiting C-16,17 epoxidation in 16,17-dihydro GAs or after C-13 hydroxylation. Overall, deactivation of GA(1) and GA(4) just below the shoot apex effectively restricts their florigenicity in Lolium and, conversely, with GA(5), C-2 and C-13 protection against deactivation allows its high florigenicity. Speculatively, such differences in GA access to the shoot apex of grasses may be important for separating floral induction from inflorescence emergence and thus could influence their survival under conditions of herbivore predation.

A circadian rhythm set by dusk determines the expression of FT homologs and the short-day photoperiodic flowering response in Pharbitis.

Seasonal control of flowering through responsiveness to daylength shows extreme variation. Different species flower in response to long days or short days (SDs), and this difference evolved several times. The molecular mechanisms conferring these responses have been compared in detail only in Arabidopsis thaliana and rice (Oryza sativa) and suggest that a conserved pathway confers daylength responses through regulation of FLOWERING LOCUS T (FT) transcription by CONSTANS (CO). We studied Pharbitis (Ipomoea nil; formerly, Pharbitis nil), a widely used SD model species and a member of the Convolvulaceae, and showed using transgenic plants together with detailed expression analysis that two putative orthologs of FT (Pn FT1 and Pn FT2) promote flowering specifically under SDs. These genes are expressed only under SDs, and light flashes given during the night reduce their expression and prevent flowering. We demonstrate that in Pharbitis a circadian rhythm set by the light-to-dark transition at dusk regulates Pn FT expression, which rises only when the night is longer than 11 h. Furthermore, Pharbitis accessions that differ in their critical night-length responses express Pn FT at different times after dusk, demonstrating that natural genetic variation influencing the clock regulating Pn FT expression alters the flowering response. In these assays, Pn FT mRNA abundance was not related to Pn CO expression, suggesting that Pn FT may be regulated by a different transcription factor in Pharbitis. We conclude that SD response in Pharbitis is controlled by a dedicated light sensitive clock, set by dusk, that activates Pn FT transcription in darkness, a different mechanism for measuring daylength than described for Arabidopsis and rice.

Prognostic value of the 6 min walk test and self-perceived symptom severity in older patients with chronic heart failure.

BACKGROUND: The 6 min walk test (6-MWT) is a simple and popular test for evaluating functional status in patients with chronic heart failure (CHF). However, the prognostic value of the 6-MWT in a large, representative sample of CHF patients, and in patients with different degrees of left ventricular systolic dysfunction (LVSD) remains unclear. METHODS AND RESULTS: Of an initial population of 1592 patients, 212 died representing a crude death rate of 13.3%. In surviving patients, the median time to follow-up period was 36.6 months [inter-quartile range (IQR) 28-45 months]. Five variables remained independent predictors of all-cause mortality; decreasing 6-MWT distance, self-perceived signs of breathlessness at night (SOBAN), beta-blocker usage, elevated log NT-proBNP, and reduced haemoglobin concentration. We also dichotomized our analysis by LVSD status (mild LVSD). For patients with >mild LVSD, 6-MWT remained an important prognostic indicator but not in patients with mild LVSD. The 6-MWT provides little prognostic utility in patients with

Changes in body composition in patients with left ventricular systolic dysfunction initiated on beta-blocker therapy.

BACKGROUND: Increasing body mass index, cholesterol and body fat are associated with a better prognosis in patients with left ventricular systolic dysfunction (LVSD). Beta-blocker usage is associated with changes in body composition and increased body fat. The present study investigated 12-month changes in body composition in patients with LVSD initiated on beta-blocker therapy. METHODS: The relation between beta-blocker use and body composition was evaluated in 91 patients (75% male) with LVSD. Body composition was assessed by bioelectrical impedance. RESULTS: Seventeen patients died during the study period. There was no statistical difference among beta-blocker usage, beta-blocker type, or changes in body fat, basal metabolic rate, impedance, fat-free mass, fat mass and total body water. There were no significant differences between any of these measures and beta-blocker usage. CONCLUSION: After 12 months, changes in body composition were not found to be influenced by initiation of beta-blocker therapy in patients with LVSD.

Regulation of flowering in the long-day grass Lolium temulentum by gibberellins and the FLOWERING LOCUS T gene.

Seasonal control of flowering often involves leaf sensing of daylength coupled to time measurement and generation and transport of florigenic signals to the shoot apex. We show that transmitted signals in the grass Lolium temulentum may include gibberellins (GAs) and the FLOWERING LOCUS T (FT) gene. Within 2 h of starting a florally inductive long day (LD), expression of a 20-oxidase GA biosynthetic gene increases in the leaf; its product, GA(20), then increases 5.7-fold versus short day; its substrate, GA(19), decreases equivalently; and a bioactive product, GA(5), increases 4-fold. A link between flowering, LD, GAs, and GA biosynthesis is shown in three ways: (1) applied GA(19) became florigenic on exposure to LD; (2) expression of LtGA20ox1, an important GA biosynthetic gene, increased in a florally effective LD involving incandescent lamps, but not with noninductive fluorescent lamps; and (3) paclobutrazol, an inhibitor of an early step of GA biosynthesis, blocked flowering, but only if applied before the LD. Expression studies of a 2-oxidase catabolic gene showed no changes favoring a GA increase. Thus, the early LD increase in leaf GA(5) biosynthesis, coupled with subsequent doubling in GA(5) content at the shoot apex, provides a substantial trail of evidence for GA(5) as a LD florigen. LD signaling may also involve transport of FT mRNA or protein because expression of LtFT and LtCONSTANS increased rapidly, substantially (>80-fold for FT), and independently of GA. However, because a LD from fluorescent lamps induced LtFT expression but not flowering, the nature of the light response of FT requires clarification.

The involvement of gibberellin 20-oxidase genes in phytochrome-regulated petiole elongation of Arabidopsis.

Long day (LD) exposure of rosette plants causes rapid stem/petiole elongation, a more vertical growth habit, and flowering; all changes are suggestive of a role for the gibberellin (GA) plant growth regulators. For Arabidopsis (Arabidopsis thaliana) L. (Heynh), we show that enhancement of petiole elongation by a far-red (FR)-rich LD is mimicked by a brief (10 min) end-of-day (EOD) FR exposure in short day (SD). The EOD response shows red (R)/FR photoreversibility and is not affected in a phytochrome (PHY) A mutant so it is mediated by PHYB and related PHYs. FR photoconversion of PHYB to an inactive form activates a signaling pathway, leading to increased GA biosynthesis. Of 10 GA biosynthetic genes, expression of the 20-oxidase, AtGA20ox2, responded most to FR (up to a 40-fold increase within 3 h). AtGA20ox1 also responded but to a lesser extent. Stimulation of petiole elongation by EOD FR is reduced in a transgenic AtGA20ox2 hairpin gene silencing line. By contrast, it was only in SD that a T-DNA insertional mutant of AtGA20ox1 (ga5-3) showed reduced response. Circadian entrainment to a daytime pattern provides an explanation for the SD expression of AtGA20ox1. Conversely, the strong EOD/LD FR responses of AtGA20ox2 may reflect its independence of circadian regulation. While FR acting via PHYB increases expression of AtGA20ox2, other GA biosynthetic genes are known to respond to R rather than FR light and/or to other PHYs. Thus, there must be different signal transduction pathways, one at least showing a positive response to active PHYB and another showing a negative response.

Flowering of the grass Lolium perenne: effects of vernalization and long days on gibberellin biosynthesis and signaling.

Almost 50 years ago, it was shown that gibberellin (GA) applications caused flowering in species normally responding to cold (vernalization) and long day (LD). The implication that GAs are involved with vernalization and LD responses is examined here with the grass Lolium perenne. This species has an obligatory requirement for exposure to both vernalization and LD for its flowering (inflorescence initiation). Specific effects of vernalization or LD on GA synthesis, content, and action have been documented using four treatment pairs: nonvernalized or vernalized plants exposed to short days (SDs) or LDs. Irrespective of vernalization status, exposure to two LDs increased expression of L. perenne GA 20-oxidase-1 (LpGA20ox1), a critical GA biosynthetic gene, with endogenous GAs increasing by up to 5-fold in leaf and shoot. In parallel, LD led to degradation of a DELLA protein, SLENDER (within 48 h of LD or within 2 h of GA application). There was no effect on GA catabolism or abscisic acid content. Loss of SLENDER, which is a repressor of GA signaling, confirms the physiological relevance of increased GA content in LD. For flowering, applied GA replaced the need for LD but not that for vernalization. Thus, GAs may be an LD, leaf-sourced hormonal signal for flowering of L. perenne. By contrast, vernalization had little impact on GA or SLENDER levels or on SLENDER degradation following GA application. Thus, although vernalization and GA are both required for flowering of L. perenne, GA signaling is independent of vernalization that apparently impacts on unrelated processes.

Gibberellin structure and function: biological activity and competitive inhibition of gibberellin 2- and 3-oxidases.

Some gibberellin (GA) analogues, especially with C-16,17 modifications of GA(5), can inhibit growth of plants apparently by acting as competitors with the endogenous substrate of GA biosynthetic enzymes. Here, we directly confirm the competitive action of GA derivatives but also show that some analogues may retain significant bioactivity. A recombinant 3-oxidase from pea, which converts GA(20) to bioactive GA(1), was inhibited by GA(5), and 16,17-dihydro-GA(5) derivatives, especially if the C-17 alkyl chain length was increased by up to three carbons or if the C-13 hydroxyl was acetylated. Genetic confirmation that GA(5) analogues target 3-oxidases in vivo was provided by comparing the growth response of a WT (LE) pea with a 3-oxidase mutant (le-1). Two pea 2-oxidases that inactivate bioactive GAs, were inhibited by GA(1) and GA(3) but were generally insensitive to GA(5) analogues. alpha-Amylase production by barley half-seeds in response to GA analogues provided a method to study their action when effects on GA biosynthesis were excluded. This bioactivity assay showed that 16,17-dihydro GA(5) analogues have some inherent activity but mostly less than for GA(5) (5-50-fold), which in turn was 100-fold less active than GA(1) and GA(3). However, although C-17 alkyl derivatives with one or two added carbons showed little bioactivity and were purely 3-oxidase inhibitors, adding a third carbon (the 17-n-propyl-16,17-dihydro GA(5) analogue) restored bioactivity to that of GA(5). Furthermore, this analogue has lost its capacity to inhibit stem elongation of Lolium temulentum (Mander et al., Phytochemistry 49:1509-1515, 1998a), although it strongly inhibits the 3-oxidase. Thus, the effectiveness of a GA derivative as a growth retardant will reflect the balance between its bioactivity and its capacity to inhibit the terminal enzyme of GA biosynthesis. The weaker growth inhibition in dicots including pea (approximately 10%) than in monocots such as L. temulentum (>35%) is suggestive of taxonomic differences in the bioactivity of GAs and/or their effects on GA biosynthesis.

Gibberellins and flowering of grasses and cereals: prizing open the lid of the "florigen" black box.

Comprehensive studies in grasses show that gibberellins (GAs) play a role as a florigen. For Lolium temulentum, which flowers in response to a single long day (LD), GAs are a transmitted signal, their content increasing in the leaf early in the LD and then, hours later, at the shoot apex. There is a continuous trail of evidence of hormonal action of these GAs for L. temulentum and support for a similar role in the flowering of other LD-responsive temperate grasses and cereals. A characteristic of the initial flowering responses of grasses and cereals is their limited stem elongation. Interestingly, it is GAs with low effectiveness for stem elongation, GA5 and GA6, that reach the shoot apex and, structurally, are probably not degraded by 2-oxidase enzymes. By contrast, GA1 and GA4 cause stem elongation, may be inactive for floral evocation, and do not reach the vegetative shoot apex apparently because of susceptibility to degradation. However, GA4 can be florally active if protected against 2-oxidases either structurally or by using a 2-oxidase inhibitor. Later in inflorescence development, GA1 and GA4 can be detected at the shoot apex and are florally active if applied. The 2-oxidase restricting accessibility to the apex has probably declined at this time so there is a second florigenic, LD-regulated GA action. A growing body of molecular evidence supporting these actions of GA may provide a future basis for manipulating flowering of grasses and cereals.

Synthesis of gibberellin GA6 and its role in flowering of Lolium temulentum.

The induction of flowering by one long day (LD) in the grass Lolium temulentum is most closely mimicked by application of the gibberellins (GAs) GA(5) or GA(6), both of which occur naturally. These gibberellins promote floral development but have little effect on stem elongation. Endogenous GA(5) and GA(6) contents in the shoot apex double on the day after the LD and, for GA(5) (and we presume for GA(6) as well) reach a concentration known to be inductive for the excised shoot apex in vitro. They are, therefore, strong candidates as LD floral stimuli in this grass. The synthesis of GA(6) and an examination of its florigenic properties in L. temulentum are described.

Effects of 17-alkyl-16,17-dihydrogibberellin A(5) derivatives on growth and flowering in Lolium temulentum.

Several gibberellins in which the 16-methyl group of the 16-epimers of dihydro-GA(5) had been replaced by ethyl, n-propyl and n-butyl were prepared and tested at doses of 1, 5 or 25&mgr;g per plant for their effects on stem growth and flowering of the grass Lolium temulentum. The ethyl and n-propyl derivatives were most inhibitory of elongation, the exo-isomers being more active than the endo-forms. While both isomers of dihydro-GA(5) promoted flowering, among the 17-alkyl analogues, only the exo-ethyl derivative showed significant activity.