During attempts to clone the gene for the Escherichia coli outer membrane protein III another gene was recovered. Its nucleotide sequence was determined and the deduced amino acid sequence showed that the gene does not encode protein III. It codes for a 173-residue polypeptide; 21 NH2-terminal residues are typical for a signal peptide. The sequence around the putative site (Ala-Cys) for removing this peptide, Ala-Ile-Ala-Ala-Cys-Ser-Ser-Asn, is highly homologous to that of the major cell envelope lipoprotein (Braun lipoprotein) surrounding its processing site; it is also homologous to the consensus pentapeptide Leu-Leu-Ala-Gly-Cys present in other lipoproteins of gram-negative bacteria. It could be shown that the gene expresses a lipoprotein with all the properties, including the amino acid composition, of the peptidoglycan-associated lipoprotein (PAL) [Mizuno, T. (1979) J. Biochem. (Tokyo) 86, 991-1000]. Therefore, the cloned gene is the pal gene. The protein does not contain hydrophobic regions which would serve as a membrane anchor. Tandemly repeated amino acid sequences exist at and near the NH2-terminus of the mature protein which are homologous to such repeats in the Braun lipoprotein, suggesting a common origin of this part of the two proteins. Attempts to place a transposon into the pal gene were unsuccessful. Hence the complete absence of the protein may be lethal and its function remains unknown.

OBJECTIVE

Obesity and hypertension, known pro-inflammatory states, are identified determinants for increased retinal microvascular abnormalities. However, the molecular link between inflammation and microvascular degeneration remains elusive. Thioredoxin-interacting protein (TXNIP) is recognised as an activator of the NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome. This study aims to examine TXNIP expression and elucidate its role in endothelial inflammasome activation and retinal lesions.

METHODS

Spontaneously hypertensive (SHR) and control Wistar (W) rats were compared with groups fed a high-fat diet (HFD) (W+F and SHR+F) for 8-10 weeks.

RESULTS

Compared with W controls, HFD alone or in combination with hypertension significantly induced formation of acellular capillaries, a hallmark of retinal ischaemic lesions. These effects were accompanied by significant increases in lipid peroxidation, nitrotyrosine and expression of TXNIP, nuclear factor κB, TNF-α and IL-1β. HFD significantly increased interaction of TXNIP-NLRP3 and expression of cleaved caspase-1 and cleaved IL-1β. Immunolocalisation studies identified TXNIP expression within astrocytes and Müller cells surrounding retinal endothelial cells. To model HFD in vitro, human retinal endothelial (HRE) cells were stimulated with 400 μmol/l palmitate coupled to BSA (Pal-BSA). Pal-BSA triggered expression of TXNIP and its interaction with NLRP3, resulting in activation of caspase-1 and IL-1β in HRE cells. Silencing Txnip expression in HRE cells abolished Pal-BSA-mediated cleaved IL-1β release into medium and cell death, evident by decreases in cleaved caspase-3 expression and the proportion of live to dead cells.

CONCLUSIONS

These findings provide the first evidence for enhanced TXNIP expression in hypertension and HFD-induced retinal oxidative/inflammatory response and suggest that TXNIP is required for HFD-mediated activation of the NLRP3 inflammasome and the release of IL-1β in endothelial cells.

OBJECTIVE

To audit current UK practice of the management of severe sepsis in children against the 2002 American College of Critical Care Medicine/Pediatric Advanced Life Support (ACCM-PALS) guideline.

METHODS

Prospective observational study.

METHODS

17 UK paediatric intensive care units (PICUs) and two UK PICU transport services.

METHODS

200 children accepted for PICU admission within 12 h of arrival in hospital, whether or not successfully transported to a PICU, with a discharge diagnosis of sepsis or suspected sepsis.

METHODS

Medical interventions, physiological and laboratory data to determine the presence or absence of shock, inter-hospital transfer times, predicted mortality (using the Paediatric Index of Mortality, version 2 (PIM2) scoring system) and observed mortality.

RESULTS

34/200 (17%) children died following referral. Although children defined as being in shock received significantly more fluid (p<0.001) than those who were not in shock, overall fluid and inotrope management suggested by the 2002 ACCM-PALS guideline was not followed in 62% of shocked children. Binary logistic regression analysis demonstrated that the odds ratio for death, if shock was present at PICU admission, was 3.8 (95% CI 1.4 to 10.2, p = 0.008).

CONCLUSIONS

The presence of shock at PICU admission is associated with an increased risk of death. Despite clear consensus guidelines for the emergency management of children with severe sepsis and septic shock, most children received inadequate fluid resuscitation and inotropic support in the crucial few hours following presentation.

An Arabidopsis cDNA clone encoding 4-coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism, was identified and sequenced. The predicted amino acid sequence is similar to those of other cloned 4CL genes. Southern blot analysis indicated that 4CL is single-copy gene in Arabidopsis. Northern blots showed that 4CL expression was activated early during seedling development. The onset of 4CL expression was correlated with the onset of lignin deposition in cotyledons and roots 2-3 days after germination. The timing of the expression of a parsley 4CL1-GUS fusion in transgenic Arabidopsis seedlings was examined in parallel and was very similar to that of endogenous 4CL. In mature plants, highest 4CL expression was observed in bolting stems, where relatively large amounts of lignin accumulate. Both 4CL and 4CL1-GUS mRNA accumulation was strongly and transiently activated by wounding of mature Arabidopsis leaves. 4CL expression was specifically activated within 6 h after infiltration of Arabidopsis ecotype Columbia leaves with a Pseudomonas syringae pv. maculicola strain harboring the bacterial avirulence gene avrB, which causes in incompatible interaction. The timing of 4CL activation was identical to the previously observed activation of PAL gene expression in this interaction. No activation of 4CL expression was observed in a compatible interaction caused by a Pseudomonas syringae pv. maculicola strain without avrB.

OBJECTIVE

Although cardiac dysfunction plays an important role in the pathogenesis of sepsis, the mechanisms that underlie cardiac dysfunction in sepsis remain poorly understood. Bacterial peptidoglycan-associated lipoprotein (PAL), an outer-membrane protein of Gram-negative bacteria, was recently found to be released into the bloodstream in sepsis and to cause inflammation and death in mice. The present studies assessed the effects of PAL on cardiomyocyte function and its signal transduction in cardiomyocytes.

METHODS

Randomized prospective animal study.

METHODS

Research laboratory.

METHODS

Male C57BL/6 mice, B6;129S-Tnfrsf1a(tm1Imx) Tnfrsf1b(tm1Imx)/J knockout mice, Toll-like receptor 2 (TLR2) knockout mice, and myeloid differentiation factor 88 (MyD88) knockout mice.

METHODS

None.

RESULTS

Immunohistochemical staining and immunoblot analysis indicated that intravenously injected PAL bound to myocardium. Injection of PAL decreased cardiac function in vivo. Challenge with PAL altered cell shortening and Ca2+ transients in isolated mouse cardiomyocytes but not in cardiomyocytes isolated from TLR2 -/- and MyD88 -/- mice. Cytokine profiling arrays demonstrated that tumor necrosis factor-alpha (TNFalpha), granulocyte colony-stimulating factor, and interferon-gamma-production were elevated in PAL-treated cardiomyocytes. Increased TNFalpha production was abolished in MyD88 -/- cardiomyocytes but restored by adenovirally mediated expression of MyD88. PAL did not affect cell shortening and Ca2+ cycling in cardiomyocytes obtained from mice deficient for TNFalpha receptor (TNFR) 1 and TNFR2 (TNFR1/2 -/-).

CONCLUSIONS

Our data reveal that PAL uses the TLR2/MyD88 signaling cascade to induce cardiomyocyte dysfunction and inflammatory responses and that TNFalpha is a major mediator of PAL-induced dysfunction in cardiomyocytes. These studies suggest that circulating PAL and other TLR2 agonists may contribute to cardiac dysfunction in sepsis.

Pal and Cpl-1, two purified bacteriophage lytic enzymes, were tested for their in vitro activity, alone and in combination, against several serotypes of Streptococcus pneumoniae, including penicillin-resistant strains. The enzymes demonstrated synergism in their ability to cleave the bacterial peptidoglycan and thus may be more efficient for the prevention and elimination of pneumococcal colonization.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics containing the characteristic thioether cross-links lanthionine and methyllanthionine. To date, no analogues of lantibiotics that contain nonproteinogenic amino acids have been reported. In this study, in vitro-reconstituted lacticin 481 synthetase was used in conjunction with synthetic peptide substrates containing nonproteinogenic amino acids to generate 11 analogues of lacticin 481. These analogues contained sarcosine and aminocyclopropanoic acid in place of Gly5, D-valine at position 6, 4-cyanoaminobutyric acid in place of Glu13, beta(3)-homoarginine at the position of Asn15, N-butylglycine and beta-Ala at Met16, naphthylalanine (Nal) at Trp19, 4-pyridynylalanine (Pal) at Phe21, and homophenylalanine (hPhe) at Phe23. Of these analogues, the Trp19Nal and Phe23hPhe mutants provided zones of inhibition larger than the parent compound in agar diffusion assays against the indicator strains Lactococcus lactis HP and Bacillus subtilis 6633. These two compounds also demonstrated improved MIC values against liquid cultures of L. lactis HP.

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

Much has been written about the benefits and applications of Peer Assisted Learning (PAL) in the literature. Curriculum developers increasingly consider PAL as a vehicle to help undergraduate healthcare students learn to teach; an outcome which has received more attention in the UK since the General Medical Council stated in Tomorrow's Doctors that medical graduates must 'Be able to demonstrate appropriate teaching skills'. This guide is primarily designed to assist curriculum developers, course organisers and educational researchers develop and implement their own PAL initiatives. It is structured around a PAL planning framework consisting of 24 questions. The questions are grouped in threes, around eight themes. Each question is discussed with reference to the PAL literature and other related subjects, and is exemplified by responses from a recent PAL project developed at The University of Edinburgh. Working through the 24 questions, particularly with discussion in a small planning group, will enable readers to efficiently develop their ideas for PAL into comprehensive and practical project plans cognisant of current educational theory, existing PAL literature and the local context. The framework is particularly suitable for those who want to develop healthcare undergraduate PAL initiatives yet have little or no experience of PAL, as it provides an introduction to the relevant literature field and a step-by-step process for the design and implementation of such projects. It will also be of interest to those with experience of PAL and those seeking a structured framework for planning non-PAL curriculum developments in undergraduate healthcare curricula.

Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase are important enzymes in allocating significant amounts of carbon from phenylalanine into the biosynthesis of several important secondary metabolites. Tea is an important crop of commerce known for its beverage and medicinally important flavonoid compounds, mainly catechins. As metabolic flux for the operation of the flavonoid pathway is maintained through the activities of PAL and C4H, thus, catechins biosynthesis in tea is critically dependent on the products of these enzymes. We examined the expression of PAL and C4H. Sequence encoding CsPAL was isolated from tea by polymerase chain reaction using sequence information available at the NCBI GenBank. Sequence encoding C4H was isolated from tea by using differential display of mRNA and rapid amplification of cDNA ends technology. CsC4H (AY641731) comprised of 1,352 bp full-length cDNA with open reading frame of 1,173 bp encoding 390 amino acids. Catechin contents decreased in response to drought stress (DS), abscisic acid (ABA), and gibberellic acid (GA(3)) treatments but increased in response to wounding. The expression of CsPAL and CsC4H showed the same behavior under the above treatments and was also in accordance with the catechin contents. A positive correlation between catechin contents and gene expression suggested a critical role of the enzymes in catechins biosynthesis and a crosstalk between phenylpropanoid and flavonoid pathways.

The genomic context and phylogenetic distribution of the tol-pal gene cluster and homologues to its various components have been investigated. The structure of this operon is well conserved across the gram negative bacteria, and the machine encoded by these genes probably evolved with the appearance of gram negative bacteria. Since the evolutionary appearance of the operon some species appear to have lost the genes. These bacteria seem to fall into two classes, namely obligate intracellular parasites and bacteria that produce large numbers of outer membrane vesicles. The evolution of the alphabeta and gamma proteobacteria was accompanied by the association of an additional gene (ybgC) with the operon. Several coincidences of genomic context argue for an important role of the tol-pal operon in cell envelope maintenance. Genes homologous to tolQ and tolR proved to be very widespread being found throughout the eubacteria, and one example in the archea, this distribution argues for an ancient origin of these genes. The genomic context of these genes often suggests a role in micronutrient uptake. Interestingly in all the cases examined the tolQ and tolR genes or their homologues appear to be present as a pair, with a potential for a tight translational regulation.

Co-factor homeodomain proteins such as Drosophila Homothorax (Hth) and Extradenticle (Exd) and their respective vertebrate homologs, the Meis/Prep and Pbx proteins, can increase the DNA-binding specificity of Hox protein transcription factors and appear to be required for many of their developmental functions. We show that the unc-62 gene encodes the C. elegans ortholog of Hth, and that maternal-effect unc-62 mutations can cause severe posterior disorganization during embryogenesis (Nob phenotype), superficially similar to that seen in embryos lacking function of either the two posterior-group Hox genes nob-1 and php-3 or the caudal homolog pal-1. Other zygotically acting unc-62 alleles cause earlier embryonic arrest or incompletely penetrant larval lethality with variable morphogenetic defects among the survivors, suggesting that unc-62 functions are required at several stages of development. The differential accumulation of four unc-62 transcripts is consistent with multiple functions. The C. elegans exd homologs ceh-20 and ceh-40 interact genetically with unc-62 and may have overlapping roles in embryogenesis: neither CEH-20 nor CEH-40 appears to be required when the other is present, but loss of both functions causes incompletely penetrant embryonic lethality in the presence of unc-62(+) and complete embryonic lethality in the presence of an unc-62 hypomorphic allele.

We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans. INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment. INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively. To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses. The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases. These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.

OBJECTIVE

The aim of this study was to assess the potential therapeutic role of para-aortic lymphadenectomy (PAL) in high-risk patients with endometrial cancer.

METHODS

We studied two groups of patients with endometrial cancer who underwent operation at Mayo Clinic (Rochester, MN) during the interval 1984 to 1993: (1) 137 patients at high risk for para-aortic lymph node involvement (myometrial invasion >50%, palpable positive pelvic nodes, or positive adnexae), excluding stage IV disease, and (2) 51 patients with positive nodes (pelvic or para-aortic), excluding stage IV disease. By our definition, PAL required removal of five or more para-aortic nodes.

RESULTS

In both groups, no significant difference existed between patients who had PAL (PAL+) and those who did not (PAL-) in regard to clinical or pathologic variables, percentage irradiated, or surgical or radiation complications. Among the 137 high-risk patients, the 5-year progression-free survival was 62% and the 5-year overall survival was 71% for the PAL- group compared with 77 and 85%, respectively, for the PAL+ group (P = 0.12 and 0.06, respectively). For the 51 patients with positive nodes, the 5-year progression-free survival and 5-year overall survival for the PAL- group were 36 and 42% compared with 76 and 77% for the PAL+ group (P = 0.02 and 0.05, respectively). Lymph node recurrences were detected in 37% of the PAL- patients but in none of the PAL+ patients (P = 0.01). Multivariate analysis suggested that submission to PAL was a cogent predictor of progression-free survival (odds ratio = 0.25; P = 0.01) and overall survival (odds ratio = 0.23; P = 0.006).

CONCLUSIONS

These results suggest a potential therapeutic role for formal PAL in endometrial cancer.

Body mass index (BMI) in conjunction with indices of energy turnover, e.g. physical activity levels (PAL), was recently proposed for classifying adult chronic energy deficiency (CED). Three deprived populations in Africa and Asia were chosen to assess the classification system. Repeated monitoring showed that the combined indicator was affected by instability in PALs and produced implausible discontinuities in the prevalence of different grades of CED. The use of BMI alone produced coherent data with changing BMI distributions, and only 5% of the population would have been wrongly classified as being malnourished, because of being thin but active. The risk of misclassification would be even smaller for populations with BMI distribution shifted towards the right. The prevalence of CED was consistently related within each country to indices of socio-economic status. Yet in Zimbabwe 18% of women and 6% of men had Grade I obesity compared with 11% and 14%, respectively, with CED. Less than 1% Indian and Ethiopian adults were obese but 61% of women and 70% of men were classified as CED in India and 57% and 50%, respectively, in Ethiopia. We propose that adult BMI alone is sufficient to provide important new insights into the problems of food availability and its control in less developed countries.

The TolQ, TolR, TolA, TolB, and Pal proteins appear to function in maintaining the integrity of the outer membrane, as well as facilitating the uptake of the group A colicins and the DNA of the infecting filamentous bacteriophages. Sequence data showed that these genes are clustered in a 6-kb segment of DNA with the gene order orf1 tolQ tolR tolA tolB pal orf2 (a newly identified open reading frame encoding a 29-kD9 protein). Like those containing orf1, bacteria containing an insertion mutation in this gene showed no obvious phenotype. Analysis of beta-galactosidase activity from fusion constructs in which the lac operon was fused to various genes in the cluster showed that the genes in this region constitute two separate operons: orf1 tolQRA and tolB pal orf2. In the orf1 tolQRA operon, translation of MR was dependent on translation of the upstream tolQ region. Consistent with this result, no functional ribosome-binding site for TolR synthesis was detected.

The first three-dimensional structure of phenylalanine ammonia lyase (PAL) has been determined at 2.1 A resolution for PAL from Rhodosporidium toruloides. The enzyme is structurally similar to the mechanistically related histidine ammonia lyase (HAL), with PAL having an additional approximately 160 residues extending from the common fold. We propose that catalysis (including lowering the pK(a) of nonacidic C3 of l-phenylalanine for an E1cb mechanism) is potentially governed by dipole moments of seven alpha helices associated with the PAL active site (six positive poles and one negative pole). Cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) resides atop the positive poles of three helices, for increasing its electrophilicity. The helix dipoles appear fully compatible with a model of phenylalanine docked in the active site of PAL having the first covalent bond formed between the amino group of substrate and the methylidene group of MIO: 12 highly conserved residues (near the N termini of helices for enhancing function) are poised to serve roles in substrate recognition, MIO activation, product separation, proton donation, or polarizing electrons from the phenyl ring of substrate for activation of C3; and a highly conserved His residue (near the C terminus of the one helix that directs its negative pole toward the active site to increase the residue's basicity) is positioned to act as a general base, abstracting the pro-S hydrogen from C3 of substrate. A similar mechanism is proposed for HAL, which has a similar disposition of seven alpha helices and similar active-site residues. The helix dipoles appear incompatible with a proposed mechanism that invokes a carbocation intermediate.

OBJECTIVE

Pyothorax-associated lymphoma (PAL) is a non-Hodgkin's lymphoma developing in the pleural cavity after a long-standing history of pyothorax. Full details of PAL are provided here.

METHODS

Clinical and pathologic findings were reviewed in 106 patients with PAL collected through a nationwide survey in Japan.

RESULTS

Age of the patients with PAL was 46 to 82 years (median, 64 years), with a male/female ratio of 12.3:1. All patients had a 20- to 64-year (median, 37-year) history of pyothorax resulting from artificial pneumothorax for treatment of pulmonary tuberculosis (80%) or tuberculous pleuritis (17%). The most common symptoms on admission were chest and/or back pain (57%) and fever (43%). Laboratory data showed that the serum neuron-specific enolase level was occasionally elevated (3.55 to 168.7 ng/mL; median, 18.65 ng/mL), suggesting a possible diagnosis of small-cell lung cancer. Histologically, PAL usually showed a diffuse proliferation of large cells of B-cell type (88%). In situ hybridization study showed that PAL in 70% of the patients was Epstein-Barr virus (EBV)-positive. PAL was responsive to chemotherapy, but the overall prognosis was poor, with a 5-year survival of 21.6%.

CONCLUSIONS

This study established the distinct nature of PAL as a disease entity. PAL is a non-Hodgkin's lymphoma of exclusively B-cell phenotype in the pleural cavity of patients with long-standing history of pyothorax, and is strongly associated with EBV infection. Development of PAL is closely related to antecedent chronic inflammatory condition; therefore, PAL should be defined as malignant lymphoma developing in chronic inflammation.

BACKGROUND

The pioneering ancestor of land plants that conquered terrestrial habitats around 500 million years ago had to face dramatic stresses including UV radiation, desiccation, and microbial attack. This drove a number of adaptations, among which the emergence of the phenylpropanoid pathway was crucial, leading to essential compounds such as flavonoids and lignin. However, the origin of this specific land plant secondary metabolism has not been clarified.

RESULTS

We have performed an extensive analysis of the taxonomic distribution and phylogeny of Phenylalanine Ammonia Lyase (PAL), which catalyses the first and essential step of the general phenylpropanoid pathway, leading from phenylalanine to p-Coumaric acid and p-Coumaroyl-CoA, the entry points of the flavonoids and lignin routes. We obtained robust evidence that the ancestor of land plants acquired a PAL via horizontal gene transfer (HGT) during symbioses with soil bacteria and fungi that are known to have established very early during the first steps of land colonization. This horizontally acquired PAL represented then the basis for further development of the phenylpropanoid pathway and plant radiation on terrestrial environments.

CONCLUSIONS

Our results highlight a possible crucial role of HGT from soil bacteria in the path leading to land colonization by plants and their subsequent evolution. The few functional characterizations of sediment/soil bacterial PAL (production of secondary metabolites with powerful antimicrobial activity or production of pigments) suggest that the initial advantage of this horizontally acquired PAL in the ancestor of land plants might have been either defense against an already developed microbial community and/or protection against UV.

OBJECTIVE

To validate a two-question questionnaire on physical activity with the doubly labeled water (DLW) method.

METHODS

Cross-sectional study.

METHODS

Nine volunteers, age 33-75 years, with a mean body mass index (BMI) (kg m(-2)) of 27.4.

METHODS

A questionnaire with one question on physical activity at work and one question on physical activity during leisure time. The answers were converted into a PAL (physical activity level=energy expenditure/basal metabolic rate) value, which was validated with the DLW method.

RESULTS

The mean values (s.d.) of PAL for the questionnaire and DLW measurements were 1.7 (0.1) and 1.7 (0.1), respectively, with a mean difference of 0.004 (0.172).

CONCLUSIONS

The results were promising to the extent that they could be used in large-scale epidemiological studies.

Hypermethylation of the FMR1 promoter reduces its transcriptional activity, resulting in the mental retardation and macroorchidism characteristic of Fragile X syndrome. How exactly methylation causes transcriptional silencing is not known but is relevant if current attempts to reactivate the gene are to be successful. Understanding the effect of methylation requires a better understanding of the factors responsible for FMR1 gene expression. To this end we have identified five evolutionarily conserved transcription factor binding sites in this promoter and shown that four of them are important for transcriptional activity in neuronally derived cells. We have also shown that USF1, USF2, and alpha-Pal/Nrf-1 are the major transcription factors that bind the promoter in brain and testis extracts and suggest that elevated levels of these factors account in part for elevated FMR1 expression in these organs. We also show that methylation abolishes alpha-Pal/Nrf-1 binding to the promoter and affects binding of USF1 and USF2 to a lesser degree. Methylation may therefore inhibit FMR1 transcription not only by recruiting histone deacetylases but also by blocking transcription factor binding. This suggests that for efficient reactivation of the FMR1 promoter, significant demethylation must occur and that current approaches to gene reactivation using histone deacetylase inhibitors alone may therefore have limited effect.

Systemic acquired resistance is an important component of the disease-resistance arsenal of plants, and is associated with an enhanced potency for activating local defense responses upon pathogen attack. Here we demonstrate that pretreatment with benzothiadiazole (BTH), a synthetic activator of acquired resistance in plants, augmented the sensitivity for low-dose elicitation of coumarin phytoalexin secretion by cultured parsley (Petroselinum crispum L.) cells. Enhanced coumarin secretion was associated with potentiated activation of genes encoding Phe ammonia-lyase (PAL). The augmentation of PAL gene induction was proportional to the length of pretreatment with BTH, indicating time-dependent priming of the cells. In contrast to the PAL genes, those for anionic peroxidase were directly induced by BTH in the absence of elicitor, thus confirming a dual role for BTH in the activation of plant defenses. Strikingly, the ability of various chemicals to enhance plant disease resistance correlated with their capability to potentiate parsley PAL gene elicitation, emphasizing an important role for defense response potentiation in acquired plant disease resistance.

OBJECTIVE

The objective of this work is to perform a systematic review and meta-analysis of all randomized controlled trials comparing a single intravenous dose of palonosetron (PAL) 0.25 mg with other 5-HT(3)R in patients receiving moderately or highly emetogenic (MoHE) chemotherapy.

METHODS

Several databases were searched, including MEDLINE, EMBASE, LILACS, and CENTRAL. The primary endpoints were the incidence of acute and delayed nausea and vomiting. The side effects of each treatment were analyzed. A subgroup analysis was performed to evaluate the influence of the use of corticosteroids. The results are expressed as risk ratio (RR) and the correspondent 95% confidence interval (CI).

RESULTS

Five studies were included, with 2057 patients. PAL was compared with ondansetron, granisetron, and dolasetron. Patients in PAL group had less nausea, both acute (RR = 0.86; CI 95% = 0.76 to 0.96; p = 0.007) and delayed (RR = 0.82; CI95% = 0.75 to 0.89; p < 0.00001). They also had less acute vomiting (RR = 0.76; CI 95% = 0.66 to 0.88; p = 0.0002) and delayed vomiting (RR = 0.76; CI95% = 0.68 to 0.85; p < 0.00001). There were no statistical differences in side effects like headache (RR = 0.84; CI 95% = 0.61 to 1.17; p = 0.30), dizziness (RR = 0.40; CI 95% = 0.13 to 1.27; p = 0.12), constipation (RR = 1.29; CI 95% = 0.77 to 2.17; p = 0.33) or diarrhea (RR = 0.67; CI 95% = 0.24 to 1.85; p = 0.44). Patients receiving PAL presented less nausea and vomiting regardless of the use of corticoids. We found no statistical heterogeneity in the global analysis.

CONCLUSIONS

PAL was more effective than the other 5-HT(3)R in preventing acute and delayed CINV in patients receiving MoHE treatments, regardless of the use of concomitant corticosteroids.

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues.