The incompatible pathosystem between resistant cotton (Gossypium barbadense cv. 7124) and Verticillium dahliae strain V991 was used to study the cotton transcriptome changes after pathogen inoculation by RNA-Seq. Of 32,774 genes detected by mapping the tags to assembly cotton contigs, 3442 defence-responsive genes were identified. Gene cluster analyses and functional assignments of differentially expressed genes indicated a significant transcriptional complexity. Quantitative real-time PCR (qPCR) was performed on selected genes with different expression levels and functional assignments to demonstrate the utility of RNA-Seq for gene expression profiles during the cotton defence response. Detailed elucidation of responses of leucine-rich repeat receptor-like kinases (LRR-RLKs), phytohormone signalling-related genes, and transcription factors described the interplay of signals that allowed the plant to fine-tune defence responses. On the basis of global gene regulation of phenylpropanoid metabolism-related genes, phenylpropanoid metabolism was deduced to be involved in the cotton defence response. A closer look at the expression of these genes, enzyme activity, and lignin levels revealed differences between resistant and susceptible cotton plants. Both types of plants showed an increased level of expression of lignin synthesis-related genes and increased phenylalanine-ammonia lyase (PAL) and peroxidase (POD) enzyme activity after inoculation with V. dahliae, but the increase was greater and faster in the resistant line. Histochemical analysis of lignin revealed that the resistant cotton not only retains its vascular structure, but also accumulates high levels of lignin. Furthermore, quantitative analysis demonstrated increased lignification and cross-linking of lignin in resistant cotton stems. Overall, a critical role for lignin was believed to contribute to the resistance of cotton to disease.

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (<em>PAL</em>), is encoded by four genes in Arabidopsis thaliana. Whereas <em>PAL</em> function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis <em>pal</em>1 and <em>pal</em>2 single mutants and with limited phenotypic alterations in the <em>pal</em>1 <em>pal</em>2 double mutant, significant modifications occur in the transcriptome and metabolome of the <em>pal</em> mutants. The disruption of <em>PAL</em> led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of <em>PAL</em>1 and <em>PAL</em>2 are delineated, and <em>PAL</em>1 is qualified as being more important for the generation of phenylpropanoids.

BACKGROUND

Docosahexaenoic acid (DHA) plays an important role in neural function. Decreases in plasma DHA are associated with cognitive decline in healthy elderly adults and in patients with Alzheimer's disease. Higher DHA intake is inversely correlated with relative risk of Alzheimer's disease. The potential benefits of DHA supplementation in age-related cognitive decline (ARCD) have not been fully examined.

OBJECTIVE

Determine effects of DHA administration on improving cognitive functions in healthy older adults with ARCD.

METHODS

Randomized, double-blind, placebo-controlled, clinical study was conducted at 19 U.S. clinical sites. A total of 485 healthy subjects, aged ≥55 with Mini-Mental State Examination >26 and a Logical Memory (Wechsler Memory Scale III) baseline score ≥1 standard deviation below younger adults, were randomly assigned to 900 mg/d of DHA orally or matching placebo for 24 weeks. The primary outcome was the CANTAB Paired Associate Learning (PAL), a visuospatial learning and episodic memory test.

RESULTS

Intention-to-treat analysis demonstrated significantly fewer PAL six pattern errors with DHA versus placebo at 24 weeks (difference score, -1.63 ± 0.76 [-3.1, -0.14, 95% CI], P = .03). DHA supplementation was also associated with improved immediate and delayed Verbal Recognition Memory scores (P < .02), but not working memory or executive function tests. Plasma DHA levels doubled and correlated with improved PAL scores (P < .02) in the DHA group. DHA was well tolerated with no reported treatment-related serious adverse events.

CONCLUSIONS

Twenty-four week supplementation with 900 mg/d DHA improved learning and memory function in ARCD and is a beneficial supplement that supports cognitive health with aging.

BACKGROUND

Clinicaltrials.gov, Identifier: NCT0027813.

Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (<em>PAL</em>1)-[beta]-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The <em>PAL</em>1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-staining procedure was developed to distinguish between GUS activity and fungal structures. The <em>PAL</em>1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific <em>PAL</em> inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced <em>PAL</em>1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific for the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced <em>PAL</em>1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the <em>PAL</em>1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility.

The marginal zone of the spleen forms an intriguing area in which a variety of cell types are combined. Several of these cell types seem to have a fixed position in the marginal zone, such as the marginal zone macrophages, the marginal metallophilic macrophages at the inner border, and, to a lesser extent, the marginal zone B cells. For other cell types--T lymphocytes, small B cells, and dendritic cells--the marginal zone is only a temporary residence. It is this combination of relatively sessile cell populations and the continuous influx and passing of bloodborne immunocompetent cells that turn the marginal zone into a dynamic area, particularly apt for antigen processing and recognition. In no other lymphoid organ can such a unique combination of cells and functions be found. The opening of the arterial blood stream in the marginal sinuses results in a reduction of the velocity of the blood stream, and antigens are initially screened in the marginal zone. To this, extremely potent phagocytic cells, the marginal zone macrophages, are present which can take up and phagocytize large foreign particles, such as bacteria and effete red blood cells. Further filtration of the blood takes place in the filtration beds of the red pulp. The marginal zone macrophages express membrane receptors for bacterial polysaccharides which lead to efficient phagocytosis, probably even in the absence of prior opsonization. Antigenic fragments produced this way can be taken up by dendritic cells that enter the spleen by the blood as part of a mobile surveillance immune system. Dendritic cells present antigen to T cells in the outer area of the T cell-dependent PALS, leading to clustering and enrichment of antigen-specific T cells. Antigens in the marginal zone can also directly associate with memory B cells thought to reside here for longer times, having intimate contact with the marginal zone macrophages. B memory cells then migrate into the PALS and present antigen to T cells. The marginal zone therefore functions not only as an area of initial filtration and phagocytosis of antigens from the blood, but also as a site of lymphocyte emigration. Some of the incoming T and B lymphocytes in the recirculating pool enter the white pulp from the marginal zone. The underlying force and selective molecular mechanisms that guide this migration are unknown. Both B and T lymphocytes recirculate through the outer PALS area on their way to the follicles and the inner PALS, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

This study aimed at developing a shortened version of the EORTC QLQ-C30, one of the most widely used health-related quality of life questionnaires in oncology, for palliative care research. The study included interviews with 41 patients and 66 health care professionals in palliative care to determine the appropriateness, relevance and importance of the various domains of the QLQ-C30. Item response theory methods were used to shorten scales. Patients and health care professionals rated pain, physical function, emotional function, fatigue, global health status/quality of life, nausea/vomiting, appetite, dyspnoea, constipation, and sleep as most important. Therefore, these scales/items were retained in the questionnaire. Four scales were shortened without reducing measurement precision. Important dimensions not covered by the questionnaire were identified. The resulting 15-item EORTC QLQ-C15-PAL is a 'core questionnaire' for palliative care. Depending on the research questions, it may be supplemented by additional items, modules or questionnaires.

Phenylalanine ammonia-lyase (<em>PAL</em>) is encoded by a small family of genes in Arabidopsis. We cloned and partially characterized one of these genes, <em>PAL</em>1. The deduced amino acid sequence is highly similar to <em>PAL</em> from bean, parsley, and rice. The promoter contains sequence elements homologous to two putative regulatory elements conserved among several phenylpropanoid genes. The regulation of the <em>PAL</em>1 gene was examined by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing <em>PAL</em>1-GUS gene fusions. The <em>PAL</em>1 promoter was activated early in seedling development and in adult plants was strongly expressed in the vascular tissues of roots and leaves, but was not active in the root tip or the shoot apical meristem. In flowers, expression was observed in sepals, anthers, and carpels, but not in petals. Transcripts encoded by the endogenous <em>PAL</em> genes and GUS transcripts from the <em>PAL</em>1-GUS gene fusion were induced by wounding, HgCl2-stress, and light. Analysis of the regulatory properties of 5' deleted promoters showed that the proximal region of the promoter to -290 was sufficient to establish the full tissue-specific pattern of expression and that the proximal region to -540 was responsive to environmental stimuli. Negative and positive elements were located between -1816 and -823 and between -823 and -290, respectively.

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95-Discs Large-zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (<em>Pals</em>)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets <em>Pals</em>1 to tight junctions by binding to a PDZ domain protein, <em>Pals</em>1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with <em>Pals</em>1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the <em>Pals</em>1 PDZ domain. In agreement, we find that a Drosophila homologue of <em>Pals</em>1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila <em>Pals</em>1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity.

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.

OBJECTIVE

To describe average levels of free-living energy expenditure in people from affluent societies and to determine the influence of body weight, height, age and sex.

METHODS

Analysis of 574 measurements of total energy expenditure (TEE, assessed by the doubly-labelled water method); basal metabolic rate (BMR, directly measured or derived from similar directly measured proxy measures such as during sleep); activity energy expenditure (AEE, derived as TEE-BMR); and physical activity level (PAL, derived as TEE/BMR) from people aged 2-95 years. The dataset was extracted from 1614 published and unpublished measurements in 1156 subjects after exclusion of repeat estimates and subjects in special physiological or behavioural states (eg pregnancy, athletic or military training etc).

RESULTS

A separate analysis of data from non-ambulant subjects, and from elite endurance athletes (all excluded from the main dataset) established the limits of human daily energy expenditure at around 1.2 x BMR and 4.5 x BMR. In the main analysis, the validity of PAL as an index of TEE adjusted for BMR was tested and confirmed. Regression equations were then derived to describe TEE, BMR, AEE and PAL in terms of body weight, height, age and sex. As anticipated, TEE, BMR and AEE were all positively related to weight and height, while age was a negative predictor, especially of activity. The influence of weight disappeared when TEE was expressed as PAL, but height and age remained as highly significant predictors. For all three components, females expended 11% less energy on average than males after adjustment for weight, height and age. Average levels of energy expenditure in different age and sex groups are tabulated.

CONCLUSIONS

There now exists a large and robust database of energy expenditure measurements obtained by the doubly-labelled water method. Analysis of the data from affluent societies shows that, in general, levels of energy expenditure are similar to the recommendations for energy requirements adopted by FAO/WHO/UNU (1985) and UK Department of Health (1991). PAL values for active subjects tend to be higher than is currently assumed. The current analysis provides a substantial body of normal data against which other estimates can be compared.

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.

OBJECTIVE

Experimental and clinical studies of septic shock support the concept that early resuscitation with fluid and inotropic therapies improves survival in a time-dependent manner. The new American College of Critical Care Medicine-Pediatric Advanced Life Support (ACCM-PALS) Guidelines for hemodynamic support of newborns and children in septic shock recommend this therapeutic approach. The objective of this study was to determine whether early septic shock reversal and use of resuscitation practice consistent with the new ACCM-PALS Guidelines by community physicians is associated with improved outcome.

METHODS

A 9-year (January 1993-December 2001) retrospective cohort study was conducted of 91 infants and children who presented to local community hospitals with septic shock and required transport to Children's Hospital of Pittsburgh. Shock reversal (defined by return of normal systolic blood pressure and capillary refill time), resuscitation practice concurrence with ACCM-PALS Guidelines, and hospital mortality were measured.

RESULTS

Overall, 26 (29%) patients died. Community physicians successfully achieved shock reversal in 24 (26%) patients at a median time of 75 minutes (when the transport team arrived at the patient's bedside), which was associated with 96% survival and >9-fold increased odds of survival (9.49 [1.07-83.89]). Each additional hour of persistent shock was associated with >2-fold increased odds of mortality (2.29 [1.19-4.44]). Nonsurvivors, compared with survivors, were treated with more inotropic therapies (dopamine/dobutamine [42% vs 20%] and epinephrine/norepinephrine [42% vs 6%]) but not increased fluid therapy (median volume; 32.9 mL/kg vs 20.0 mL/kg). Resuscitation practice was consistent with ACCM-PALS Guidelines in only 27 (30%) patients; however, when practice was in agreement with guideline recommendations, a lower mortality was observed (8% vs 38%).

CONCLUSIONS

Early recognition and aggressive resuscitation of pediatric-neonatal septic shock by community physicians can save lives. Educational programs that promote ACCM-PALS recommended rapid, stepwise escalations in fluid as well as inotropic therapies may have value in improving outcomes in these children.

As a step toward a comprehensive description of lignin biosynthesis in Populus trichocarpa, we identified from the genome sequence 95 phenylpropanoid gene models in 10 protein families encoding enzymes for monolignol biosynthesis. Transcript abundance was determined for all 95 genes in xylem, leaf, shoot and phloem using quantitative real-time PCR (qRT-PCR). We identified 23 genes that most probably encode monolignol biosynthesis enzymes during wood formation. Transcripts for 18 of the 23 are abundant and specific to differentiating xylem. We found evidence suggesting functional redundancy at the transcript level for phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT) and coniferyl aldehyde 5-hydroxylase (CAld5H). We carried out an enumeration-based motif identification and discriminant analysis on the promoters of all 95 genes. Five core motifs correctly discriminate the 18 xylem-specific genes from the 77 non-xylem genes. These motifs are similar to promoter elements known to regulate phenylpropanoid gene expression. This work suggests that genes in monolignol biosynthesis are regulated by multiple motifs, often related in sequence.

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.

Vascular endothelial growth factor receptor-3 (VEGFR-3) is essential for embryonic cardiovascular development, but thereafter becomes confined to the lymphatic endothelium in adult tissues. We have here studied VEGFR-3 expression in experimental wounds of pigs and chronic inflammatory wounds of humans. In healing incisional and punch biopsy wounds made in the dorsal skin of pigs, angiogenic blood vessels, identified by use of the blood vascular endothelial markers vWF and PAL-E and the basal lamina protein laminin, developed into the granulation tissue stroma from day 4 onward, being most abundant on days 5 and 6 and regressing thereafter. VEGFR-3-positive vessels were observed in the granulation tissue from day 5 onward. These vessels were distinct from the PAL-E/laminin/vWF-positive vessels and fewer in number, and they appeared to sprout from pre-existing VEGFR-3-positive lymphatic vessels at the wound edge. Unlike the blood vessels, very few VEGFR-3-positive lymphatic vessels persisted on day 9 and none on day 14. In chronic wounds such as ulcers and decubitus wounds of the lower extremity of humans, VEGFR-3 was also weakly expressed in the vascular endothelium. Our results suggest that transient lymphangiogenesis occurs in parallel with angiogenesis in healing wounds and that VEGFR-3 becomes up-regulated in blood vessel endothelium in chronic inflammatory wounds.

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (<em>PAL</em>) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana <em>PAL</em> genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana <em>PAL</em> genes (<em>PAL</em>1 and <em>PAL</em>2) are structurally similar to <em>PAL</em> genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of <em>PAL</em>1 and <em>PAL</em>2. Expression of <em>PAL</em>1 and <em>PAL</em>2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana <em>PAL</em> gene, <em>PAL</em>3, differs significantly from <em>PAL</em>1 and <em>PAL</em>2 and other sequenced plant <em>PAL</em> genes. <em>PAL</em>3 contains an additional intron, and its deduced amino acid sequence is less homologous to other <em>PAL</em> proteins. The <em>PAL</em>3 promoter region lacks several sequence motifs conserved between A. thaliana <em>PAL</em>1 and <em>PAL</em>2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana <em>PAL</em>3 was expressed at very low levels under the conditions examined.

The pal locus of Antirrhinum majus was cloned using the transposable element, Tam 3, as a probe. The pal clone was used to examine, at the molecular level, those aspects of instability previously observed phenotypically and genetically. The effects of temperature and of genetic background on excision of the element at pal are considered, and related quantitatively to the phenotype. We describe the identification of the transcript of the pal locus and show that insertion of Tam 3 blocks the production of a normal pal transcript in developing flower buds.

The reduced form of glutathione (GSH), when supplied to suspension cultured cells of bean (Phaseolus vulgaris L.) at concentrations in the range 0.01 to 1.0 millimolar, stimulates transcription of defense genes including those that encode cell wall hydroxyproline-rich glycoproteins and the phenylpropanoid biosynthetic enzymes phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) involved in lignin (PAL) and phytoalexin (PAL, CHS) production. Transcriptional activation of these genes leads to marked accumulation of the corresponding transcripts, contributing to a massive change in the overall pattern of protein synthesis which closely resembles that previously observed in response to fungal elicitor. GSH causes a marked increase in extractable PAL activity, whereas the oxidized form of glutathione, constituent amino acids, or other reducing agents are inactive. Possible roles of GSH in signaling biological stress are discussed.

BACKGROUND

The role of speckle tracking in the assessment of left atrial (LA) deformation dynamics is not established. We sought to determine the feasibility and reference ranges of LA longitudinal strain indices measured by speckle tracking in a population of normal subjects.

METHODS

In 60 healthy individuals, peak atrial longitudinal strain (PALS) and time to peak longitudinal strain (TPLS) were measured using a 12-segment model for the left atrium. Values were obtained by averaging all segments (global PALS and TPLS) and by separately averaging segments measured in the two apical views (4- and 2-chamber average PALS and TPLS).

RESULTS

Adequate tracking quality was achieved in 97% of segments analyzed. Inter and intra-observer variability coefficients of measurements ranged between 2.9% and 5.4%. Global PALS was 42.2 +/- 6.1% (5-95 degrees percentile range 32.2-53.2%), and global TPLS was 368 +/- 30 ms (5-95 degrees percentile range 323-430 ms). The 2-chamber average PALS was slightly higher than the 4-chamber average PALS (44.3 +/- 6.0% vs 40.1 +/- 7.9%, p < 0.0001), whereas no differences in TPLS were found (p = 0.93).

CONCLUSIONS

Speckle tracking is a feasible technique for the assessment of longitudinal myocardial LA deformation. Reference ranges of strain indices were reported.

Biosynthesis of phenylpropanoid natural products in tobacco was perturbed by introduction of a heterologous (bean) phenylalanine ammonia-lyase (PAL; L-phenylalanine ammonia-lyase, EC 4.3.1.5) gene, modified by inclusion of cauliflower mosaic virus 35S enhancer sequences in its promoter. These transgenic plants can exhibit a series of unusual phenotypes including localized fluorescent lesions, altered leaf shape and texture, reduced signification in xylem, stunted growth, reduced pollen viability, and altered flower morphology and pigmentation. Genetic analysis of a transformant with severe symptoms showed that symptom development was inherited as a single, partially dominant trait and cosegregated with reduced levels of PAL activity and soluble phenylpropanoid products. Accumulation of transcripts encoded by the endogenous tobacco PAL genes was suppressed. We conclude that the transgene disrupts PAL regulation and that some of the phenotypes reflect interference with putative signals dependent on phenylpropanoid biosynthesis.

We describe a complete gene family encoding phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in one particular plant species. In parsley (Petroselinum crispum), the PAL gene family comprises two closely related members, PALPALPALPALPALPALPALPALPALPALPAL gene family members, in contrast to a high degree of coordination in the overall expression of the PAL, C4H, and 4CL genes. The only significant sequence similarities shared by all four PAL gene promoters are a TATA-proximal set of three putative cis-acting elements (boxes P, A, and L). None of these elements alone, or the promoter region containing all of them together, conferred elicitor or light responsiveness on a reporter gene in transient expression assays. The elements appear to be necessary but not sufficient for elicitor- or light-mediated PAL gene activation, similar to the situation previously reported for 4CL.

BACKGROUND

The ACCM/PALS guidelines address early correction of paediatric septic shock using conventional measures. In the evolution of these recommendations, indirect measures of the balance between systemic oxygen delivery and demands using central venous or superior vena cava oxygen saturation (ScvO(2)>> or = 70%) in a goal-directed approach have been added. However, while these additional goal-directed endpoints are based on evidence-based adult studies, the extrapolation to the paediatric patient remains unvalidated.

OBJECTIVE

The purpose of this study was to compare treatment according to ACCM/PALS guidelines, performed with and without ScvO(2) goal-directed therapy, on the morbidity and mortality rate of children with severe sepsis and septic shock. DESIGN, PARTICIPANTS AND INTERVENTIONS: Children and adolescents with severe sepsis or fluid-refractory septic shock were randomly assigned to ACCM/PALS with or without ScvO(2) goal-directed resuscitation.

METHODS

Twenty-eight-day mortality was the primary endpoint.

RESULTS

Of the 102 enrolled patients, 51 received ACCM/PALS with ScvO(2) goal-directed therapy and 51 received ACCM/PALS without ScvO(2) goal-directed therapy. ScvO(2) goal-directed therapy resulted in less mortality (28-day mortality 11.8% vs. 39.2%, p=0.002), and fewer new organ dysfunctions (p=0.03). ScvO(2) goal-directed therapy resulted in more crystalloid (28 (20-40) vs. 5 (0-20 ml/kg, p<0.0001), blood transfusion (45.1% vs. 15.7%, p=0.002) and inotropic (29.4% vs. 7.8%, p=0.01) support in the first 6 h.

CONCLUSIONS

This study supports the current ACCM/PALS guidelines. Goal-directed therapy using the endpoint of a ScvO(2>> or =70% has a significant and additive impact on the outcome of children and adolescents with septic shock.

T-B cell interactions have a central role in the development of antibody responses. Upon activation, T helper (Th) cells express the ligand for CD40, gp39, which is essential for Th cell-dependent B cell activation. The cytokines produced by activated Th cells have a regulatory role in B cell differentiation. In this study, we investigated, using immunohistochemical techniques, the in vivo time course and localization of gp39 expression and cytokine production in relation to the specific antibody production. Both the immunization with keyhole limpet hemocyanin (KLH), a thymus-dependent (TD) antigen, and trinitrophenyl (TNP)-Ficoll, a thymus-independent type 2 (TI-2) antigen, induced Th cells to express gp39. The expression of gp39 was restricted to Th cells in the outer periarteriolar lymphocyte sheaths (outer-PALS) and around the terminal arterioles (TA). Incidentally, gp39+ Th cells were found in the corona of follicles, whereas gp39+ cells were never found in the germinal centers or marginal zones of the spleen. Maximum frequencies of gp39+ cells were observed 3 and 4 d after primary and secondary immunization with KLH. After injection of TNP-Ficoll, a marked increase in gp39+ cells was observed, confirming previous observations that activated T cells are involved in TI-2 antibody responses. Analysis of the in vivo cytokine production revealed that interleukin 2 (IL-2)-, IL-4- and interferon gamma (IFN-gamma)-producing cells (IFN-gamma-PC) developed according to similar kinetics as observed for gp39+ cells. IL-2-PC and IL-4-PC were present in higher frequencies as were IFN-gamma-PC in the immune response against TNP-KLH. Double staining experiments revealed gp39+ Th cells producing IL-2, IL-4, or IFN-gamma, suggesting that these cells were involved in both the initial activation as well as the differentiation process of B cells into antibody-forming cells. Dual immunohistochemical analysis revealed gp39+ T cells and cytokine-PC in close proximity to antigen-specific, antibody-forming B cells. In conclusion, this study shows that in vivo gp39 is expressed on activated Th cells after immunization with TD and TI-2 antigens. Furthermore, the time course and compartmentalization of gp39+ expression, cytokine production and antibody formation after immunization suggest that cognate T-B cell interactions and T cell-regulated B cell differentiation occur in the outer-PALS and around the TA of the spleen.

Programmed cell death is increasingly viewed as a key component of the hypersensitive disease resistance response of plants. The generation of reactive oxygen species (ROS) such as H2O2 triggers a cell death programme in Arabidopsis suspension cultures following challenge with the bacterial elicitor harpin. Both harpin and exogenous H2O2 initiate a cell death pathway that requires gene expression, and also act as signalling molecules to induce the expression of plant defence genes encoding enzymes such as phenylalanine ammonia-lyase (<em>PAL</em>), glutathione S-transferase (GST) and anthranilate synthase (ASA1), an enzyme of phytoalexin biosynthesis in Arabidopsis. H2O2 induces the expression of <em>PAL</em>1 and GST but not that of ASA1. Harpin initiates two signalling pathways, one leading to increased ROS generation and expression of <em>PAL</em>1 and GST mRNA, and another leading to increased GST and ASA1 expression, independent of H2O2.