We used a number of receptor antagonists to determine which receptors mediate the effect of arginine vasopressin (AVP) and oxytocin (OT) on insulin release. We found that OT (10(-7) M) and AVP (10(-8) M) increased insulin release from the perfused rat pancreas with similar magnitude. The antagonist with potent V1b receptor-blocking activity, dP[Tyr(Me)2]AVP (10(-7) M), abolished the effect of OT and AVP, whereas the highly selective OT receptor antagonist L-366,948 (10(-6) M) did not change the effect of OT, nor did a V1a receptor antagonist, d(CH2)5[Tyr(Me)2]AVP (10(-7) M), change the effect of AVP. The insulin-releasing potency of OT was estimated as 9-fold less than that of AVP in RINm5F cells. Selected AVP and OT antagonists were used to study their antagonism on AVP- and OT-induced insulin release from RINm5F cells, and the order of potencies of antagonists was estimated as dP[Tyr(Me)2]AVP>> d(CH2)5[D-Phe2,Ile4]AVP>> SR-49059>> d(CH2)5[Tyr(Me)2]AVP>> desGly9d(CH2)5[Tyr(Et)2]VAVP (WK-3-6) approximately L-366,948. These results were consistent with the V1b receptor antagonistic activities of the antagonists. d[D-3-Pal]VP, a V1b receptor agonist, increased insulin release dose dependently (10(-9) to 10(-6) M), and this effect was antagonized by dP[Tyr(Me)2]AVP but not by WK-3-6 (10(-6) M). These results suggested that the stimulatory effect of both OT and AVP on insulin release from beta-cells may be mediated by V1b, but not by V1a or OT receptors.

The purpose of this study was to examine and monetize the educational outcomes of students with ADHD. Data were examined from the Pittsburgh ADHD Longitudinal Study (PALS), a follow-up study of children diagnosed with ADHD in childhood and recontacted for follow-up in adolescence and young adulthood. A comprehensive educational history was obtained for all participants from Kindergarten through 12th grade. Annual economic impact was derived from costs incurred through special education placement, grade retention, and disciplinary incidents. Results indicated that, as compared to students without ADHD, students with ADHD incurred a higher annual cost to the U.S. Education system. Specifically, a student with ADHD incurred an average annual incremental cost to society of $5,007, as compared to $318 for students in the comparison group. These results suggest that prevention and intervention strategies are greatly needed to offset the large financial impact of educating youth with ADHD.

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3' end of the psi encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3' end of the pal within psi with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.

The purpose of the current study was to test the ability of adolescents and young adults with childhood ADHD to reliably self-report delinquency history. Data were examined from the Pittsburgh ADHD Longitudinal Study (PALS), a follow-up study of children diagnosed with ADHD between 1987 and 1996. Self-report of lifetime delinquency history was compared to concurrent parent-report and to self-report 1 year later. Participants included 313 male probands and 209 demographically similar comparison individuals without ADHD. Results indicated that adolescents and young adults with childhood ADHD were more likely than comparison participants to fail to report delinquent acts reported by a parent and to recant acts they endorsed 1 year earlier. This trend was most apparent for acts of mild to moderate severity. After controlling for several covariates, current ADHD symptom severity and parent-report of the participant's tendency to lie predicted reporting fewer delinquent acts than one's parent. Current ADHD symptom severity also predicted more recanting of previously endorsed acts. Based on these findings, several recommendations are made for the assessment of delinquency history in adolescents and young adults with childhood ADHD.

BACKGROUND

Patients with obsessive compulsive disorder (OCD) demonstrate impaired cognition in some selected domains. Although serotoninergic dysfunction has been implicated in OCD, recent evidence suggests that dopamine may play a role as well.

OBJECTIVE

The aim of the study was to evaluate learning and working memory in OCD and to determine the effects of dopaminergic manipulations on these capacities.

METHODS

Visuospatial associative memory and spatial and verbal working memory were examined in 18 nondepressed patients with OCD and 18 matched healthy controls. The study further investigated whether acute administration of dopamine D2/D3 receptor agonist and antagonist would differentially modulate cognition in OCD. Each participant underwent the cognitive battery three times in a randomized double-blind, placebo-controlled crossover design.

RESULTS

Significant impairments in patients compared with controls were noted on the Cambridge Neuropsychological Test Automated Battery (CANTAB) paired associates learning (PAL) and a measure of sustained attention (rapid visual information processing, RVIP) that persisted across all sessions, with deficient strategy in the CANTAB spatial working memory task in the first session alone. Although the dopamine D2/D3 agonist, pramipexole, led to poorer performance on the PAL and RVIP tasks, no differential effects were noted between the two groups. No significant effects were noted for the D2/D3 antagonist, amisulpride.

CONCLUSIONS

The results are consistent with a specific associative memory deficit in OCD that remained robust despite possible practice effects and compensatory strategies and point to abnormal medial temporal lobe involvement in OCD in addition to the previously implicated frontostriatal loops, with no clear evidence of D2 receptor mediation.

We studied the relationship between biosynthesis of salicylates, the main chemical defenses in willow and growth of Salix pentandra by cultivating plants in the presence of 2-aminoindan-2-phosphonic acid (AIP), a powerful inhibitor of phenylalanine ammonia-lyase (PAL: EC 4.3.1.5.). AIP inhibited efficiently, though not totally, the endogenous synthesis of salicylates. This inhibition markedly increased plant growth. Exogenous application of the precursors of salicylates, benzoic acid (BA), salicylic acid (SA), and helicin, increased the levels of several individual salicylates, but decreased the growth of plants cultivated in the presence of AIP. These results suggest a trade-off between plant growth and the synthesis of salicylates in S. pentandra. Phenylalanine, which accumulated in response to inhibitor treatment, but was decreased by precursor treatments, may be a common and limiting precursor for both plant growth and salicylate synthesis. The biosynthesis of salicin is suggested to proceed mainly via benzoyl-glucose, an intermediate in the synthesis of salicylic acid. Salicin is the most obvious precursor of more substituted salicylates, salicortin, acetylsalicortin, and tremulacin. In addition, we found that the salicylate pools of mature plant parts of S. pentandra were not subject to turnover, implying that the maintenance of salicylates does not demand high resources of plants, although their initial construction is costly.

OBJECTIVE

To predict risk factors of a prolonged air leak following pulmonary lobectomy.

METHODS

From January 2003 to March 2004, 134 patients (95 male, mean age 66+/-7.7 years) underwent pulmonary lobectomy for lung tumor. Prolonged air leak (PAL) was defined as an air leak lasting 7 days or more of postoperative chest tube drainage. Chronic obstructive pulmonary disease (COPD) was defined as an FEV1 of <70% predicted and an FEV1/FVC ratio <70% based on the results of a preoperative pulmonary function test.

RESULTS

PAL occurred in 13 (9.7%) patients and it lasted 10.4+/-3.2 days. Eleven out of 13 patients with PAL were identified with this co-morbidity only. There was not reoperation for PAL. Differences in age, gender, exposure to neoadjuvant chemotherapy, body mass index (BMI) and type of lobectomy were not statistically significant. COPD remained the only variable predicted for PAL (P<0.05).

CONCLUSIONS

Prolonged air leak is quite a common pulmonary complication after pulmonary lobectomy and can be treated conservatively. This complication significantly prolongs the length of hospitalization (P<0.01). We also conclude that COPD increases the risk of an air leak persisting for longer than 7 days.

BACKGROUND

Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) deficiency that results in phenylalanine (Phe) accumulation. Pegvaliase, PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL), converts Phe to trans-cinnamic acid and ammonia, and is a potential enzyme substitution therapy to lower blood Phe in adults with PKU.

METHODS

Two Phase 3 studies, PRISM-1 and PRISM-2, evaluated the efficacy and safety of pegvaliase treatment using an induction, titration, and maintenance dosing regimen in adults with PKU. In PRISM-1, pegvaliase-naïve participants with blood Phe >600 μmol/L were randomized 1:1 to a maintenance dose of 20 mg/day or 40 mg/day of pegvaliase. Participants in PRISM-1 continued pegvaliase treatment in PRISM-2, a 4-part clinical trial that includes an ongoing, open-label, long-term extension study of pegvaliase doses of 5 mg/day to 60 mg/day.

RESULTS

Of 261 participants who received pegvaliase treatment, 72.0% and 32.6% reached ≥12 months and ≥ 24 months of study treatment, respectively, and 65% are still actively receiving treatment. Mean (SD) blood Phe was 1232.7 (386.4) μmol/L at baseline, 564.5 (531.2) μmol/L at 12 months, and 311.4 (427) μmol/L at 24 months, a decrease from baseline of 51.1% and 68.7%, respectively. Within 24 months, 68.4% of participants achieved blood Phe ≤600 μmol/L, 60.7% of participants achieved blood Phe ≤360 μmol/L, below the upper limit recommended in the American College of Medical Genetics and Genomics PKU management guidelines, and 51.2% achieved blood Phe ≤120 μmol/L, below the upper limit of normal in the unaffected population. Improvements in neuropsychiatric outcomes were associated with reductions in blood Phe and were sustained with long-term pegvaliase treatment. Adverse events (AEs) were more frequent in the first 6 months of exposure (early treatment phase) than after 6 months of exposure (late treatment phase); 99% of AEs were mild or moderate in severity and 96% resolved without dose interruption or reduction. The most common AEs were arthralgia (70.5%), injection-site reaction (62.1%), injection-site erythema (47.9%), and headache (47.1%). Acute systemic hypersensitivity events consistent with clinical National Institute of Allergy and Infectious Diseases and the Food Allergy and Anaphylaxis Network anaphylaxis criteria were observed in 12 participants (17 events); of these, 6 participants remained on treatment. Acute systemic hypersensitivity events including potential events of anaphylaxis were not associated with immunoglobulin E, and all events resolved without sequelae.

CONCLUSIONS

Results from the PRISM Phase 3 program support the efficacy of pegvaliase for the treatment of adults with PKU, with a manageable safety profile in most participants. The PRISM-2 extension study will continue to assess the long-term effects of pegvaliase treatment.

Since soluble silicon (Si) has been shown to alleviate copper (Cu) toxicity in Arabidopsis thaliana, the expression of genes involved in responses to Cu toxicity was examined by quantitative reverse transcription-polymerase chain reaction. Expression levels of three metallothionein (MT) genes were increased under Cu stress conditions whereas Cu-stressed plants treated with Si either maintained high levels or contained even higher levels of MT RNA. Cu/zinc superoxide dismutase (SOD) enzyme activity was induced by Cu toxicity. However, SOD activity was increased even more if plants were provided with extra Si and toxic levels of Cu. Previously, plants treated with elevated Cu showed increased phenylalanine ammonia lyase (<em>PAL</em>) activity that was reduced when the plants were also provided with extra Si. Since the Arabidopsis genome encodes 4 <em>PAL</em> genes (<em>PAL</em>1-4), we examined which ones were responsive to Cu and Si. <em>PAL</em> 1, <em>PAL</em> 2, and <em>PAL</em> 3 all showed similar patterns of gene expression that matched previous enzymatic data while <em>PAL</em>4 was elevated by the presence of high Cu whether Si was present or not. Taken together, these data suggested that Si permitted plants to respond to Cu toxicity more effectively and that these changes occurred at the gene expression level.

A series of investigations was undertaken to evaluate the extent and predictability of periodontal regeneration with barrier membranes in deep infrabony defects. This study reports the clinical outcomes in terms of probing attachment level (PAL) gains, probing pocket depth (PPD) reduction, and recession (REC) of marginal gingiva. Forty deep 1, 2, and 3-wall combination infrabony defects treated with membranes were evaluated 1 year postoperatively following a strict plaque control regimen (mean plaque score = 6.1%, bleeding score = 4% at 1 year). A PAL gain of 6.1 +/- 2.5 mm along with a PPD reduction of 5.9 +/- 2.5 mm were observed. A PAL gain of 2 mm or more was detected in almost 90% of the treated sites. No site lost attachment. Residual PPD was 3 mm or shallower in 95% of the sites. It is concluded that treatment of deep infrabony defects according to the principles of guided tissue regeneration and a strict plaque control regimen represents an efficacious and predictable treatment alternative.

Peptides and proteins play an important role in skin health and well-being. They are also found to contribute to skin aging and melanogenesis. Microneedles have been shown to substantially enhance skin penetration and may offer an effective means of peptide delivery enhancement. The aim of this investigation was to assess the influence of microneedles on the skin penetration of peptides using fluorescence imaging to determine skin distribution. In particular the effect of peptide chain length (3, 4, 5 amino acid chain length) on passive and MN facilitated skin penetration was investigated. Confocal laser scanning microscopy was used to image fluorescence intensity and the area of penetration of fluorescently tagged peptides. Penetration studies were conducted on excised full thickness human skin in Franz type diffusion cells for 1 and 24 hours. A 2 to 22 fold signal improvement in microneedle enhanced delivery of melanostatin, rigin and pal-KTTKS was observed. To our knowledge this is the first description of microneedle enhanced skin permeation studies on these peptides.

During the life cycles of conifer trees, such as maritime pine (Pinus pinaster Ait.), large quantities of carbon skeletons are irreversibly immobilized in the wood. In energetic terms this is an expensive process, in which carbon from photosynthesis is channelled through the shikimate pathway for the biosynthesis of phenylpropanoids. This crucial metabolic pathway is finely regulated, primarily through transcriptional control, and because phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and use should occur simultaneously. The promoters of three genes encoding the enzymes prephenate aminotransferase (PAT), phenylalanine ammonia lyase (PAL) and glutamine synthetase (GS1b) contain AC elements involved in the transcriptional activation mediated by R2R3-Myb factors. We have examined the capacity of the R2R3-Myb transcription factors Myb1, Myb4 and Myb8 to co-regulate the expression of PAT, PAL and GS1b. Only Myb8 was able to activate the transcription of the three genes. Moreover, the expression of this transcription factor is higher in lignified tissues, in which a high demand for phenylpropanoids exits. In a gain-of-function experiment, we have shown that Myb8 can specifically bind a well-conserved eight-nucleotide-long AC-II element in the promoter regions of PAT, PAL and GS1b, thereby activating their expression. Our results show that Myb8 regulates the expression of these genes involved in phenylalanine metabolism, which is required for channelling photosynthetic carbon to promote wood formation. The co-localization of PAT, PAL, GS1b and MYB8 transcripts in vascular cells further supports this conclusion.

Hepatitis C virus infection is a major health problem worldwide. Developing effective antiviral therapy for HCV is the need of the hour. The viral enzymes NS3 protease and NS5B RNA dependent RNA polymerase are essential enzymes for polyprotein processing and viral RNA replication and thus can be potential targets for screening anti-HCV compounds. A large number of phytochemicals are present in plants, which are found to be promising antiviral agents. In this study, we have screened inhibitory effect of different plant extracts against the NS3 and NS5B enzymes of hepatitis C virus. Methanolic extracts were prepared from various plant materials and their inhibitory effects on the viral enzymes were determined by in vitro enzyme assays. Effect on viral RNA replication was investigated by using TaqMan Real time RT-PCR. Interestingly, Phyllanthus amarus root (PAR) extract showed significant inhibition of HCV-NS3 protease enzyme; whereas P. amarus leaf (PAL) extract showed considerable inhibition of NS5B in the in vitro assays. Further, the PAR and PAL extracts significantly inhibited replication of HCV monocistronic replicon RNA and HCV H77S viral RNA in HCV cell culture system. However, both PAR and PAL extracts did not show cytotoxicity in Huh7 cells in the MTT assay. Furthermore, addition of PAR together with IFN-α showed additive effect in the inhibition of HCV RNA replication. Results suggest the possible molecular basis of the inhibitory activity of PA extract against HCV which would help in optimization and subsequent development of specific antiviral agent using P. amarus as potent natural source.

OBJECTIVE

The aim of the study is to evaluate the effect of moderate Sicilian red wine consumption on cardiovascular risk factors and, in particular, on some inflammatory biomarkers.

METHODS

A total of 48 subjects of both sexes who were nondrinkers or rare drinkers of moderate red wine were selected and randomly subdivided into two groups assigned to receive with a crossover design a Sicilian red wine (Nero d'Avola or Etna Torrepalino) during meals: Group A (n = 24), in whom the diet was supplemented for 4 weeks with 250 ml/day of red wine, followed by 4 weeks when they returned to their usual wine intake; and Group B (n = 24), in whom the usual wine intake was maintained for 4 weeks, followed by 4 weeks when the diet was supplemented with 250 ml/day of red wine. The following were values measured in all tests: blood glucose, total and HDL-cholesterol and triglycerides, LDL-cholesterol, LDL/HDL ratio, apolipoproteins A1 and B, Lp(a), plasma C-reactive protein, TGFbeta1, D-Dimer, Factor VII , PAl Ag, t-PA Ag, fibrinogen, oxidized LDL Ab, total plasma antioxidant capacity.

RESULTS

At the end of the red wine intake period, LDL/HDL, fibrinogen, factor VII, plasma C-reactive protein and oxidized LDL Ab were significantly decreased, while HDL-C, Apo A1,TGFbeta1, t-PA, PAI and total plasma antioxidant capacity were significantly increased.

CONCLUSIONS

Our results show a positive effect of two Sicilian red wines on many risk factors and on some inflammatory biomarkers, suggesting that a moderate consumption of red wine in the adult population is a positive component of the Mediterranean diet.

OBJECTIVE

To isolate and identify endophytic nitrogen-fixing bacteria in sugarcane growing in Cuba without chemical fertilizers.

RESULTS

Two N2-fixing isolates, 9C and T2, were obtained from surface-sterilized stems and roots, respectively, of sugarcane variety ML3-18. Both isolates showed acetylene reduction and H2 production in nitrogen-free media. Nitrogenase activity measured by H2 production was about 15 times higher for isolate 9C than for T2 or for Gluconoacetobacter diazotrophicus (PAL-5 standard strain, ATCC 49037). The nifH gene segment was amplified from both isolates using specific primers. Classification of both T2 and 9C was made on the basis of morphological, biochemical, PCR tests and 16S rDNA sequence analysis.

CONCLUSIONS

Isolate 9C was identified as a Pantoea species from its 16S rDNA, but showed considerable differences in physiological properties from previously reported species of this genus. For example, 9C can be cultured over a wide range of temperature, pH and salt concentration, and showed high H2 production (up to 67.7 nmol H2 h(-1) 10(10) cell(-1)). Isolate T2 was a strain of Gluconacetobacter diazotrophicus.

CONCLUSIONS

A new N2-fixing endophyte, i.e. Pantoea, able to produce H2 and to grow in a wide range of conditions, was isolated from sugarcane stem tissue and characterized. The strain with these attributes may well be valuable for agriculture.

OBJECTIVE

The aim of this study was to evaluate the efficacy and feasibility of the Physical Activity Leaders (PALs) program. PALs is an obesity prevention program for low-active adolescent boys from disadvantaged schools.

METHODS

The study was conducted in the Hunter Region, New South Wales (NSW), Australia from June to December 2009. Four disadvantaged secondary schools were randomized to treatment conditions for the 6-month study period. Participants were 100 adolescent boys [mean (SD) age=14.3(0.6) years; BMI=22.1 kg/m(-2) (4.6); BMI z-score=0.6 (1.2)]. The primary outcome was change in BMI and secondary outcomes were body fat (bioelectrical impedance analysis), waist circumference, muscular fitness (leg dynamometer, 90º push-up test and 7-stage sit-up test), physical activity (5-days of pedometry) and selected dietary behaviors.

RESULTS

Significant group-by-time interaction effects were found for BMI (mean difference=-0.8 kg/m(-2), p<0.001, d =0.7), BMI z-score (mean difference=-0.2, p<0.001, d=0.7), and body fat (mean difference=-1.8%, p<0.05, d=0.5), but not for waist circumference, muscular fitness or physical activity. Participants in the intervention group reduced their consumption of sugar-containing beverages.

CONCLUSIONS

The PALs program was a feasible and efficacious approach to induce healthy weight loss in adolescent boys from disadvantaged secondary schools.

Coupled radiative-convective/photochemical modeling was performed for Earth-like planets orbiting different types of stars (the Sun as a G2V, an F2V, and a K2V star). O(2) concentrations between 1 and 10(-5) times the present atmospheric level (PAL) were simulated. The results were used to calculate visible/near-IR and thermal-IR spectra, along with surface UV fluxes and relative dose rates for erythema and DNA damage. For the spectral resolution and sensitivity currently planned for the first generation of terrestrial planet detection and characterization missions, we find that O(2) should be observable remotely in the visible for atmospheres containing at least 10(-2) PAL of O(2). O(3) should be visible in the thermal-IR for atmospheres containing at least 10(-3) PAL of O(2). CH(4) is not expected to be observable in 1 PAL O(2) atmospheres like that of modern Earth, but it might be observable at thermal-IR wavelengths in "mid-Proterozoic-type" atmospheres containing approximately 10(-1) PAL of O(2). Thus, the simultaneous detection of both O(3) and CH(4) - considered to be a reliable indication of life - is within the realm of possibility. High-O(2) planets orbiting K2V and F2V stars are both better protected from surface UV radiation than is modern Earth. For the F2V case the high intrinsic UV luminosity of the star is more than offset by the much thicker ozone layer. At O(2) levels below approximately 10(-2) PAL, planets around all three types of stars are subject to high surface UV fluxes, with the F2V planet exhibiting the most biologically dangerous radiation environment. Thus, while advanced life is theoretically possible on high-O(2) planets around F stars, it is not obvious that it would evolve as it did on Earth.

Phenylalanine ammonia-lyase (PAL) is the first enzyme of phenylpropanoid biosynthesis involved in the synthesis of a multiplicity of plant natural products. We have isolated and characterized a nearly full-length cDNA clone (pmPAL-1) corresponding to a melon fruit (Cucumis melo L. var. reticulatus) gene coding for a protein which is highly similar to PAL from other plants. Melon fruit PAL is transcriptionally induced both in response to fruit ripening and wounding. PAL gene expression follows the kinetics of expression of the ethylene biosynthetic genes during fruit development. In contrast, ethylene biosynthetic genes show different induction kinetics compared to PAL expression in response to wounding. Similar results have been found for two other genes coding for enzymes involved in flavonoid biosynthesis (chalcone synthase, CHS; chalcone isomerase, CHI). Our results imply that regulation of defense gene expression in melon is a co-ordinated process in response to both ethylene and an ethylene-independent wound signal.

This study demonstrates the sequence of enhanced generation of signal molecules such as phytohormones, i.e. jasmonic acid (JA), ethylene (ET), salicylic acid (SA), and a relatively stable free radical, nitric oxide (NO), in response of Pisum sativum L. cv. Cysterski seedling leaves to the infestation of pea aphid Acyrthosiphon pisum (Harris) at a varied population size. In time from 0 to 96h after A. pisum infestation these signal molecules accumulated transiently. Moreover, the convergence of these signaling pathways occurred. JA and its methyl derivative MeJA reached the first maximum of generation at 24th hour of infestation. An increase in ET and NO generation was observed at 48th hour of infestation. The increase in SA, JA/MeJA and ET concentrations in aphid-infested leaves occurred from the 72nd to 96th hour. In parallel, an increase was demonstrated for the activities of enzymes engaged in the biosynthesis of SA, such as phenylalanine ammonia-lyase (PAL) and benzoic acid 2-hydroxylase (BA2H). Additionally, a considerable post-infestation accumulation of transcripts for PAL was observed. An increase in the activity of lipoxygenase (LOX), an important enzyme in the biosynthesis of JA was noted. This complex signaling network may contribute to the coordinated regulation of gene expression leading to specific defence responses.

BACKGROUND

Cryotherapy (the application of ice for therapeutic purposes) is one of the most common treatment modalities employed in the immediate management of acute soft tissue injury. Despite its widespread clinical use, the precise physiological responses to therapeutic cooling have not been fully elucidated, and effective evidence-based treatment protocols are yet to be established. Intermittent ice applications are thought to exert a significant analgesic effect. This could facilitate earlier therapeutic exercise after injury, potentially allowing for a quicker return to activity. The primary aim of the forthcoming study is therefore to examine the safety and effectiveness of combining intermittent ice applications with periods of therapeutic exercise in the first week after an acute ankle sprain.

METHODS

The study is a randomised controlled trial. 120 subjects with an acute grade I or grade II ankle sprain will be recruited from Accident & Emergency and a University based Sports Injury Clinic. Subjects will be randomised under strict double-blind conditions to either a standard cryotherapy (intermittent ice applications with compression) or cryokinetic treatment group (intermittent ice applications with compression and therapeutic exercise). After the first week, treatment will be standardised across groups. Assessor blinding will be maintained throughout the trial. Primary outcome will be function, assessed using the Lower Extremity Functional Scale (LEFS). Additional outcomes will include pain (10 cm Visual Analogue Scale), swelling (modified figure-of-eight method) and activity levels (activPALtrade mark physical activity monitor, PAL Technologies, Glasgow, UK). Diagnostic Ultrasound (Episcan-1-200 high frequency ultrasound scanning system, Longport International Ltd, PA) will also be used to assess the degree of soft tissue injury. After baseline assessment subjects will be followed up at 1, 2, 3 & 4 weeks post injury. All data will be analysed using repeated measures analysis of co-variance (ANCOVA).

CONCLUSIONS

This paper describes the rationale and design of a randomised controlled trial which will examine the effectiveness of two different cryotherapy protocols in the early management of acute ankle sprain.

BACKGROUND

ISRCTN13903946.

BACKGROUND

Plant secondary metabolites, including phenylpropanoids and carotenoids, are stress inducible, have important roles in potato physiology and influence the nutritional value of potatoes. The type and magnitude of environmental effects on tuber phytonutrients is unclear, especially under modern agricultural management that minimizes stress. Understanding factors that influence tuber secondary metabolism could facilitate production of more nutritious crops. Metabolite pools of over forty tuber phenylpropanoids and carotenoids, along with the expression of twenty structural genes, were measured in high-phenylpropanoid purple potatoes grown in environmentally diverse locations in North America (Alaska, Texas and Florida).

RESULTS

Phenylpropanoids, including chlorogenic acid (CGA), were higher in samples from the northern latitudes, as was the expression of phenylpropanoid genes including phenylalanine ammonia lyase (PAL), which had over a ten-fold difference in relative abundance. Phenylpropanoid gene expression appeared coordinately regulated and was well correlated with metabolite pools, except for hydroxycinnamoyl-CoA:quinatehydroxcinnamoyl transferase (HQT; r = -0.24). In silico promoter analysis identified two cis-acting elements in the HQT promoter not found in the other phenylpropanoid genes. Anthocyanins were more abundant in Alaskan samples and correlated with flavonoid genes including DFR (r = 0.91), UFGT (r = 0.94) and F3H (r = 0.77). The most abundant anthocyanin was petunidin-3-coum-rutinoside-5-glu, which ranged from 4.7 mg g-1 in Alaska to 2.3 mg g-1 in Texas. Positive correlations between tuber sucrose and anthocyanins (r = 0.85), suggested a stimulatory effect of sucrose. Smaller variation was observed in total carotenoids, but marked differences occurred in individual carotenoids, which had over a ten-fold range. Violaxanthin, lutein or zeaxanthin were the predominant carotenoids in tubers from Alaska, Texas and Florida respectively. Unlike in the phenylpropanoid pathway, poor correlations occurred between carotenoid transcripts and metabolites.

CONCLUSIONS

Analysis of tuber secondary metabolism showed interesting relationships among different metabolites in response to collective environmental influences, even under conditions that minimize stress. The variation in metabolites shows the considerable phenotypical plasticity possible with tuber secondary metabolism and raises questions about to what extent these pathways can be stimulated by environmental cues in a manner that optimizes tuber phytonutrient content while protecting yields. The differences in secondary metabolites may be sufficient to affect nutritional quality.

The grapevine (Vitis vinifera) is susceptible to many pathogens such as Botrytis cinerea, Plasmopara viticola, Erysiphe necator, and Eutypa lata. Phytochemicals are used extensively in vineyards to reduce pathogen infections, but the appearance of pesticide-resistant pathogen strains and the need for environmental protection require the use of alternative strategies. The phytohormone ethylene is assumed to play a role in the development of disease resistance. In the present study, we have treated grapevine foliar cuttings (Cabernet Sauvignon) with ethylene-releasing ethephon. This resulted in an increase in the number of pathogenesis-related protein (CHIT4c, PIN, PGIP, and GLU) gene copies and in an enhancement of phytoalexin biosynthesis by inducing the PAL and STS genes that correlated with the accumulation of stilbenes (antimicrobial compounds). Moreover, ethephon treatment triggered the protection of grapevine detached leaves and grapevine foliar cuttings against Erysiphe necator, the causal agent of powdery mildew (64% and 70%, respectively). These studies emphasize the major role of ethylene in grapevine defense.

Microspore embryogenesis (ME) is a process in which the gametophytic pollen programme of the microspore is reoriented towards a new embryo sporophytic programme. This process requires a stress treatment, usually performed in the anther or isolated microspores for several days. Despite the universal use of stress to induce ME, very few studies have addressed the physiological processes that occur in the anther during this step. To further understand the processes triggered by stress treatment, we followed the response of anthers by measuring the expression of stress-related genes in two barley (Hordeum vulgare L.) cultivars differing in their ME response. Genes encoding enzymes involved in oxidative stress (glutathione-S-transferase, GST; oxalate oxidase, OxO), in the synthesis of jasmonic acid (13-lipoxygenase, Lox; allene oxide cyclase, AOC; allene oxide synthase, AOS) and in the phenylpropanoid pathway (phenylalanine ammonia lyase, PAL), as well as those encoding PR proteins (Barwin, chitinase 2b, Chit 2b; glucanase, Gluc; basic pathogenesis-related protein 1, PR1; pathogenesis-related protein 10, PR10) were up-regulated in whole anthers upon stress treatment, indicating that anther perceives stress and reacts by triggering general plant defence mechanisms. In particular, both OxO and Chit 2b genes are good markers of anther reactivity owing to their high level of induction during the stress treatment. The effect of copper sulphate appeared to limit the expression of defence-related genes, which may be correlated with its positive effect on the yield of microspore embryos.