beta,gamma-Methylene adenosine triphosphate (AMPOPCP) has two effects on fragmented sarcoplasmic reticulum (FSR), i.e., inhibition of the rate of Ca uptake and the induction of Ca release from FSR filled with Ca. The Ca release brought about by AMPOPCP has many features in common with the mechanism of Ca-induced Ca release: i) it is inhibited by 10 mM procaine; ii) the amount of Ca release increases with increase in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the extent of saturation of FSR with Ca; iii) increase of the Ca concentration in the medium facilitates the release of Ca. However, no facilitation of Ca release upon decrease of Mg concentration in the medium is observable. AMPOPCP and caffeine potentiate each other remarkably in their Ca-releasing action, irrespective of the kind of substrate. From the mode of action of AMPOPCP on the rate of Ca uptake, the amount of phosphorylated intermediate (EP), and the effect on Sr release, it is suggested that the state of the FSR-ATP complex is crucial for Ca-induced Ca release.

The antimyotoxic and antihemorrhagic effects of Eclipta prostrata (EP) and three of its constituents (wedelolactone, WE; stigmaterol, ST; and sitosterol, SI) were investigated. The myotoxicity of crotalid venoms (Bothrops jararaca, Bothrops jararacussu and Lachesis muta), purified myotoxins (bothropstoxin, BthTX; bothropasin; and crotoxin), and polylysine was quantified in vitro by the release rate of creatine kinase (CK) from rat or mouse extensor digitorum muscles, and in vivo by the plasma CK activity in mice. The in vitro myotoxicity of the crotalid venoms and myotoxins was neutralized by simultaneous exposure of the muscles to an aqueous extract of EP or to WE. ST and SI were less effective than WE, but interacted synergistically with it. Both the EP extract and WE failed to neutralize the in vitro myotoxic effects of polylysine. The in vivo myotoxicity of venoms and myotoxins was neutralized by their preincubation with the EP extract or WE. Intravenous administration of the plant extract or WE attenuated the increase in plasma CK activity induced by subsequent intramuscular injections of the crotalid venoms or the myotoxins. EP and WE inhibited the hemorrhagic effect of B. jararaca venom, as well as the phospholipase A2 activity of crotoxin and the proteolytic activity of B. jararaca venom. The data provide direct evidence for antimyotoxic and antihemorrhagic effects of EP and WE against the crotalid venoms responsible for most cases of envenomation by snakebites in Brazil. These effects are interpreted as consequences of antiproteolytic and antiphospholipase A2 activities of EP and its constituents.

Transgenic mice that overexpress cyclooxygenase-2 (COX-2) selectively in podocytes are more susceptible to glomerular injury by adriamycin and puromycin (PAN). To investigate the potential roles of COX-2 metabolites, we studied mice with selective deletion of prostanoid receptors and generated conditionally immortalized podocyte lines from mice with either COX-2 deletion or overexpression. Podocytes that overexpressed COX-2 were virtually indistinguishable from wild-type podocytes but were significantly more sensitive to PAN-induced injury, produced more prostaglandin E(2) and thromboxane B(2), and had greater expression of prostaglandin E(2) receptor subtype 4 (EP(4)) and thromboxane receptor (TP). Treatment of COX-2-overexpressing podocytes with a TP antagonist reduced apoptosis, but treatment with an EP(4) antagonist did not. In contrast, podocytes from COX-2-knockout mice exhibited increased apoptosis, markedly decreased cell adhesion, and prominent stress fibers. In vivo, selective deletion of podocyte EP(4) did not alter the increased sensitivity to adriamycin-induced injury observed in mice overexpressing podocyte COX-2. In contrast, genetic deletion of TP in these mice prevented adriamycin-induced injury, with attenuated albuminuria and foot process effacement. These results suggest that basal COX-2 may be important for podocyte survival, but overexpression of podocyte COX-2 increases susceptibility to podocyte injury, which is mediated, in part, by activation of the thromboxane receptor.

Organisms as simple as bacteria can engage in complex collective actions, such as group motility and fruiting body formation. Some of these actions involve a division of labor, where phenotypically specialized clonal subpopulations or genetically distinct lineages cooperate with each other by performing complementary tasks. Here, we combine experimental and computational approaches to investigate potential benefits arising from division of labor during biofilm matrix production. We show that both phenotypic and genetic strategies for a division of labor can promote collective biofilm formation in the soil bacterium Bacillus subtilis. In this species, biofilm matrix consists of two major components, exopolysaccharides (EPSs) and TasA. We observed that clonal groups of B. subtilis phenotypically segregate into three subpopulations composed of matrix non-producers, EPS producers, and generalists, which produce both EPSs and TasA. This incomplete phenotypic specialization was outperformed by a genetic division of labor, where two mutants, engineered as specialists, complemented each other by exchanging EPSs and TasA. The relative fitness of the two mutants displayed a negative frequency dependence both in vitro and on plant roots, with strain frequency reaching a stable equilibrium at 30% TasA producers, corresponding exactly to the population composition where group productivity is maximized. Using individual-based modeling, we show that asymmetries in strain ratio can arise due to differences in the relative benefits that matrix compounds generate for the collective and that genetic division of labor can be favored when it breaks metabolic constraints associated with the simultaneous production of two matrix components.

UNASSIGNED

Ethyl pyruvate (EP), a potent reactive oxygen species scavenger, has been reported to contribute to the inflammatory process. However, the protective effect of ethyl pyruvate on Concanavalin A (Con A)-induced autoimmune hepatitis have not been explored. Thus, the aims of this study are to investigate both the effects of ethyl pyruvate and its mechanism of protection on Con A-induced autoimmune hepatitis in mice.

METHODS

Acute autoimmune hepatitis was induced by Con A (20 mg/kg) in Balb/C mice; ethyl pyruvate (40 mg/kg and 80 mg/kg) was administrated 1h prior to the Con A injection. At 3h, 6h and 24h post Con A injection, histological grading, proinflammatory cytokine levels and nuclear factor kappa B (NF-κB) activity were determined.

RESULTS

Following Con A challenge, cytokines TNF-α, IL-2, IL-1β and IL-6 were expressed at 3h and 6h, and the level of HMGB1 significantly increased by 24h. Pretreatment with ethyl pyruvate ameliorated the pathological effects of Con A-induced autoimmune hepatitis and significantly decreased the levels of TNF-α, IL-2, IL-6 and IL-1β at 3h and 6h and the level of HMGB1 at 6h and 24h post injection. Ethyl pyruvate blocked the degradation of IκB α and IκB β and decreased the expression of NF-κB at 24h.

CONCLUSIONS

Taken together, these results indicated that ethyl pyruvate protected against Con A-induced autoimmune hepatitis by decreasing both early (TNF-α, IL-2, IL-1β and IL-6) and late (HMGB1) cytokine expression in mice. The reduction of HMGB1 may correlate with the amelioration of NF-κB activity.

Extracellular polymeric substances (EPS) contribute to biofilm stability and adhesion properties. The EPS matrix might also be a site for free extracellular enzyme activity; however, little is known about participation of enzyme activity in EPS during biofilm formation. In this study, we analyzed the activities of beta-glucosidase, leu-aminopeptidase, and beta-glucosaminidase during the colonization of artificial substrata (glass tiles) in a stream distinguishing enzyme activity in EPS matrix (matrix-enzymes) and total biofilm extracellular enzyme activity. The 1-h incubation of a biofilm suspension and cation-exchange resin followed by centrifugation seems appropriate to extract the matrix fraction (supernatant) and measure matrix enzymes (including free and linked to EPS) in freshwater biofilms, although there is a methodological limitation for using a biofilm suspension instead of an undisrupted biofilm. Total biofilm activities and matrix-enzyme activities showed similar capabilities to decompose organic matter compounds, with a greater capacity for peptide decomposition (leu-aminopeptidase) than for polysaccharides (beta-glucosidase), and a low decomposition of chitin and peptidoglycan (beta-glucosaminidase). Matrix-enzyme activity increased with colonization time, but more slowly than that of total enzyme activity. At the beginning of the colonization experiment (days 1-4) matrix enzymes accounted for 65-81% of total biofilm enzyme activity. Higher proportion of polysaccharides in EPS versus total biofilm, and higher matrix-enzyme activities per microgram of polysaccharides in the EPS were measured during the first 1-3 days of biofilm formation, indicating a high rate of enzyme release into the matrix during this period. Relative contribution of matrix-enzyme activities decreased as biofilm matures, but was maintained at 13-37% of total enzyme activity at the 42- to 49-day-old biofilm. These enzymes, retained and conserved in the EPS, may contribute to community metabolism. When analyzing extracellular enzymes in biofilms, the contribution of matrix enzymes must be considered, especially for young biofilms.

Purified polysaccharides (EPS) prepared from the plant Echinacea purpurea are shown to strongly activate macrophages. Macrophages activated with these substances develop pronounced extracellular cytotoxicity against tumor targets. The activation is brought about by EPS alone and is independent of any cooperative effect with lymphocytes. Also the production and secretion of oxygen radicals and interleukin 1 by macrophages is increased after activation with EPS. Cells of the macrophages lineage seem to be the main target for the action of these polysaccharides. EPS has no effect on T lymphocytes. B lymphocytes show a comparatively modest proliferation after incubation with E. purpurea EPS. Thus, these compounds, which are at least in tissue culture completely nontoxic, may be suited to activate in vivo cells of the macrophage system to cytotoxicity. They may therefore be of relevance in tumor and infectious systems.

Neurotransmitter-neuroendocrine and cardiovascular responses to the administration of a psychologically stressful mixed-model test (Mental Arithmetic, Stroop Color Word Interference Task, Trier Social Stress Test) were examined in 20 male peripubertal subjects affected by anxiety disorder (group A: 14 with generalized anxiety disorder, 6 with generalized anxiety disorder and separation anxiety disorder) and 20 junior school adolescents, matched for age, without overt psychological disorders (group B). Plasma levels of norepinephrine (NE), epinephrine (EPI), adrenocorticotropic hormone (ACTH), beta-endorphin (beta-EP), cortisol (CORT), growth hormone (GH), prolactin (PRL) and testosterone (Te) were measured immediately before the beginning of the tests and 30 min later at their end. Mean prestress values of GH, PRL, beta-EP and ACTH were significantly higher in anxious subjects than in controls. There was no difference in NE, EPI, CORT and Te prestress levels in the two groups. After the psychological stress session NE, GH and Te concentrations increased significantly in anxious subjects (A), but not in controls. In contrast, beta-EP and PRL decreased significantly during the psychological stress session in anxious subjects, and were unaffected by stress in the subjects without anxiety. No significant changes were found in ACTH, CORT and EPI during the challenge either in anxious subjects or in controls, which may be attributed to the late time of poststress blood sampling. In contrast to controls, heart rate and systolic blood pressure increased significantly in anxious subjects after psychological stress testing. Our data support the hypothesis that the hyperactivity of the noradrenergic system in response to stress is associated with anxiety disorders in adolescents and might influence the responses of GH and Te. High prestress basal values of stress hormones seem to be induced in anxious subjects by the anticipation of the task or by a persistent hyperactivity of the noradrenergic system. Further studies are needed to investigate in more detail the involvement of the HPA axis in anxious adolescents by a more refined resolution of time points of blood sampling.

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coli group 1 and 4 capsules.

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

The role of fever in host defense, if indeed it has one, is poorly understood. Fever in response to exogenous agents is mediated by a host macrophage product called endogenous pyrogen (EP). Recently it has been shown that EP is probably identical to interleukin 1 (IL1), an immunostimulatory macrophage product that induces T-cell proliferation. We postulated that the pyrogenic and immunostimulatory actions of this host mediator might be interrelated and tested T-cell proliferation induced by IL1 at a temperature characteristic of fever. The T-cell proliferative response to IL1 (and to the lymphokine, interleukin 2) was greatly increased at 39 degrees C compared to 37 degrees C, while B-cell mitogenesis in response to lipopolysaccharide was not. These findings suggest that, if similar events occur in vivo, fever may have important immunoregulatory significance and call into question the current indiscriminate use of antipyretic agents.

The filamentous fungus Phoma herbarum CCFEE 5080 isolated from continental Antarctica soil was tested for exopolysaccharide (EPS) production. The fungus grew and produced EPS (up to 13.6 g/l) on a variety of carbon sources among which sorbitol was best, particularly at the concentration of 60 g/l. EPS production was maximum when the nitrogen source was NaNO3 (3 g/l) and the incubation temperature was 28 degrees C. The polysaccharide was purified by repeated precipitation in ethanol and gel filtration and characterized as a homopolymer of glucose having a molecular weight of 7.412 x 10(6); structural analysis indicated the presence of beta-1,3 and beta-1,6 linkages only. After repeated freezing and thawing of the fungal biomass in the presence of EPS, the mycelial growth was much higher than that observed after freezing in the absence of EPS and the difference increased with the number of freeze-thaw cycles. It is hypothesized that the adaptation of P. herbarum CCFEE 5080 to the Antarctic soil microclimatic conditions, characterized by low temperature, high thermal fluctuations and repeated freeze-thaw cycles, might be related to the EPS production ability.

The molecular epidemiology of group B Streptococcus (GBS) in Ireland was investigated. Invasive (n = 132) and non-invasive (n = 45) isolates, collected in 2007-2011, were analysed by multilocus locus sequence typing, capsular polysaccharide (CPS) serotyping, profiling of surface proteins, pilus islands (PI), and antimicrobial susceptibility. Isolates grouped into 45 sequence types and five main clonal complexes (CC). CC1, CC17 and CC23 represented 67.2 % of isolates and the most prevalent serotypes Ia, III and V. Serotype and surface protein genes were largely predictive of CC. Accordingly, CPS V/alp3, CPS Ib/CPS II/bca + bac, and CPS Ia/eps predominated in CC1, CC12 and CC23, respectively, and CPS III/rib in CC17 and CC19. Supporting their vaccine potential, all isolates harboured at least one PI, of which the PI-1 + PI-2a combination was most prevalent. Macrolide resistance was found in 18.6 % of isolates. erm(B) and the globally disseminated CC1/CPS V were the most common resistance mechanism and CC/CPS type, respectively. CC17, significantly associated with neonatal disease, was also prevalent in pregnant adults, but was underrepresented among non-pregnant adults. Two of 46 CC17 isolates (typically CPS III) were CPS IV. Sequence analysis confirmed capsular switching and their relatedness to CC17/CPS IV strains recently characterized in France. CPS IV, detected only in invasive isolates (6.8 %), was most prevalent in adults (12 %) and showed an increase in prevalence to that reported (1.4 %) for invasive isolates in Ireland 1997-1999. Increases in serotype IV and evidence of capsular switching in CC17 highlights the importance of ongoing surveillance of GBS and may have implications for vaccine development strategies.

OBJECTIVE

Few tools exist that provide objective accurate prediction of short-term mortality risk in patients presenting with acute heart failure (AHF). The purpose was to describe the accuracy of several biomarkers for predicting short-term death rates in patients diagnosed with AHF in the emergency department (ED).

METHODS

The Biomarkers in ACute Heart failure (BACH) trial was a prospective, 15-center, international study of patients presenting to the ED with nontraumatic dyspnea. Clinicians were blinded to all investigational markers, except troponin and natriuretic peptides, which used the local hospital reference range. For this secondary analysis, a core lab was used for all markers except troponin. This study evaluated patients diagnosed with AHF by the on-site emergency physician (EP).

RESULTS

In the 1,641 BACH patients, 466 (28.4%) had an ED diagnosis of AHF, of whom 411 (88.2%) had a final diagnosis of AHF. In the ED-diagnosed HF patients, 59% were male, 69% had a HF history, and 19 (4.1%) died within 14 days of their ED visit. The area under the curve (AUC) for the 14-day mortality receiver operating characteristic (ROC) curve was 0.484 for brain natriuretic peptide (BNP), 0.586 for N-terminal pro-B-type natriuretic peptide (NT-proBNP), 0.755 for troponin (I or T), 0.742 for adrenomedullin (MR-proADM), and 0.803 for copeptin. In combination, MR-proADM and copeptin had the best 14-day mortality prediction (AUC = 0.818), versus all other markers.

CONCLUSIONS

MR-proADM and copeptin, alone or in combination, may provide superior short-term mortality prediction compared to natriuretic peptides and troponin. Presented results are explorative due to the limited number of events, but validation in larger trials seems promising.

OBJECTIVE

Oligodeoxynucleotides containing unmethylated CpG dinucleotides induce innate and adaptive immunity through Toll-like receptor 9 (TLR9). In the present study, we have examined the ability of a novel agonist of TLR9, called immunomodulatory oligonucleotide (IMO), to enhance effects of a HER-2/neu plasmid DNA electroporation/adenovirus (DNA-EP/Ad) vaccine.

METHODS

BALB/NeuT mice were treated with DNA-EP vaccine alone, IMO alone, or the combination of two agents starting at week 13, when all mice showed mammary neoplasia. Tumor growth and survival were documented. Antibody and CD8+ T-cell responses were determined. Peptide microarray analysis of sera was carried out to identify immunoreactive epitopes. Additionally, microCT and microPET imaging was carried out in an advanced-stage tumor model starting treatment at week 17 in BALB/NeuT mice.

RESULTS

The combination of DNA-EP and IMO resulted in significant tumor regression or delay to tumor progression. 2-Deoxy-2-[18F]fluoro-D-glucose microPET and microCT imaging of mice showed reduced tumor size in the DNA-EP/IMO combination treatment group. Mice treated with the combination produced greater antibody titers with IgG2a isotype switch and antibody-dependent cellular cytotoxicity activity than did mice treated with DNA-EP vaccine. An immunogenic B-cell linear epitope, r70, within the HER-2 dimerization domain was identified through microarray analysis. Heterologous DNA-EP/Ad vaccination combined with IMO increased mice survival.

CONCLUSIONS

The combination of HER-2/neu genetic vaccine and novel agonist of TLR9 had potent antitumor activity associated with antibody isotype switch and antibody-dependent cellular cytotoxicity activities. These results support possible clinical trials of the combination of DNA-EP/Ad-based cancer vaccines and IMO.

Amyloid-beta (Abeta) peptides, generated by the proteolysis of beta-amyloid precursor protein by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta through EP(2) and EP(4) receptors, and here we have examined the molecular mechanism. Activation of EP(2) and EP(4) receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP(2), but not EP(4), receptor-mediated stimulation of the Abeta production. In contrast, inhibitors of endocytosis suppressed EP(4), but not EP(2), receptor-mediated stimulation. Activation of gamma-secretase was observed with the activation of EP(4) receptors but not EP(2) receptors. PGE(2)-dependent internalization of the EP(4) receptor was observed, and cells expressing a mutant EP(4) receptor lacking the internalization activity did not exhibit PGE(2)-stimulated production of Abeta. A physical interaction between the EP(4) receptor and PS-1, a catalytic subunit of gamma-secretases, was revealed by immunoprecipitation assays. PGE(2)-induced internalization of PS-1 and co-localization of EP(4), PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP(4) receptor null mice. These results suggest that PGE(2)-stimulated production of Abeta involves EP(4) receptor-mediated endocytosis of PS-1 followed by activation of the gamma-secretase, as well as EP(2) receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.

We reviewed the epidemiological aspects of antipsychotic-induced movement disorders as they pertain to older patients. The incidence and prevalence of drug-induced parkinsonism and tardive dyskinesia (TD) are significantly greater in the older patient than in the younger patient whereas akathisia seems to occur evenly across the age spectrum and dystonia is uncommon among older patients. The literature on risk factors associated with treatment-emergent movement disorders is highly variable. Treatment practices vary across the age range and the interaction between age and antipsychotic dosage confounds our understanding of the relative importance of treatment-related risk factors. However, there is general agreement that pre-existing extrapyramidal signs (EPS) increase the vulnerability of the patient to developing significant drug-induced movement disorders. Elderly patients with dementia are at greater risk than patients without dementia for persistent drug-induced EPS. Management of drug-induced movement disorders in the older patient requires careful consideration of the contraindications imposed by such agents as anticholinergics and beta-blockers. At present, well-controlled double-blind studies of second-generation antipsychotics such as clozapine, risperidone. olanzapine or quetiapine for reducing the risk of treatment-emergent movement disorders in the elderly have not been published. However, open-label studies of atypical antipsychotics demonstrate a markedly lower incidence of both EPS and TD compared with conventional antipsychotic treatment in the elderly. There is emerging literature in support of atypical antipsychotics for the treatment of existing drug-induced movement disorders. More controversial is the use of adjunctive antioxidants in newly treated patients who are vulnerable to drug-induced movement disorders. While the evidence is mixed in support of antioxidants for the treatment of TD, the possibility remains that prophylactic use of antioxidants may help reduce the incidence of TD. The development of a drug-induced movement disorder often reduces the quality of life in an elderly patient. Effective pharmacological management requires cooperation from the patient and family, which can be fostered early in the patient's care through proper informed consent. The risks and benefits of antipsychotic treatment in the elderly patient need to be communicated to the patient and family. At the present time, there is no consistently effective treatment for patients with TD once it develops. Therefore, attention should focus on its prevention and close monitoring.

In view of the therapeutic efficacy of adrenocorticotropic hormone (ACTH) in the treatment of infantile spasms (IS) with hypsarrhythmia, we studied the cerebrospinal fluid (CSF) levels of ACTH in 15 children (4-10 months) affected by IS with hypsarrhythmia (eight cryptogenic forms, seven secondary to perinatal distress) and in age-matched controls. Lumbar puncture was performed in all but one case before any kind of treatment. In another case, CSF was collected 3 weeks after a spontaneous remission. Both ACTH and beta-endorphin (beta-EP), the other peptide related to the same precursor (proopiomelanocortin), were measured by specific radioimmunoassay after gel chromatography. While beta-EP levels were unchanged in the two groups of patients, ACTH concentrations of cryptogenic (3.75 +/- 2.40 fmol/ml, Mean +/- SD p less than 0.05) and secondary (6.36 +/- 3.70, NS) forms were lower than in controls (10.90 +/- 5.79). On the other hand, ACTH was higher in the case studied after therapy (9.0) and in the case presenting a spontaneous clinical and EEG remission (15.0). These data indicate that in children affected by IS with hypsarrhythmia (mainly of cryptogenic type), CSF levels of ACTH are lower, while levels of beta-EP remain normal. It would therefore appear that central ACTH content may play a possible role in the pathogenesis of IS with hypsarrhythmia.

The presence, genetic identity and diversity of algal endosymbionts (Symbiodinium) in 114 species from 69 genera (20 families) of octocorals from the Great Barrier Reef (GBR), the far eastern Pacific (EP) and the Caribbean was examined, and patterns of the octocoral-algal symbiosis were compared with patterns in the host phylogeny. Genetic analyses of the zooxanthellae were based on ribosomal DNA internal transcribed spacer 1 (ITS1) region. In the GBR samples, Symbiodinium clades A and G were encountered with A and G being rare. Clade B zooxanthellae have been previously reported from a GBR octocoral, but are also rare in octocorals from this region. Symbiodinium G has so far only been found in Foraminifera, but is rare in these organisms. In the Caribbean samples, only Symbiodinium clades B and C are present. Hence, Symbiodinium diversity at the level of phylogenetic clades is lower in octocorals from the Caribbean compared to those from the GBR. However, an unprecedented level of ITS1 diversity was observed within individual colonies of some Caribbean gorgonians, implying either that these simultaneously harbour multiple strains of clade B zooxanthellae, or that ITS1 heterogeneity exists within the genomes of some zooxanthellae. Intracladal diversity based on ITS should therefore be interpreted with caution, especially in cases where no independent evidence exists to support distinctiveness, such as ecological distribution or physiological characteristics. All samples from EP are azooxanthellate. Three unrelated GBR taxa that are described in the literature as azooxanthellate (Junceella fragilis, Euplexaura nuttingi and Stereonephthya sp. 1) contain clade G zooxanthellae, and their symbiotic association with zooxanthellae was confirmed by histology. These corals are pale in colour, whereas related azooxanthellate species are brightly coloured. The evolutionary loss or gain of zooxanthellae may have altered the light sensitivity of the host tissues, requiring the animals to adopt or reduce pigmentation. Finally, we superimposed patterns of the octocoral-algal symbiosis onto a molecular phylogeny of the host. The data show that many losses/gains of endosymbiosis have occurred during the evolution of octocorals. The ancestral state (azooxanthellate or zooxanthellate) in octocorals remains unclear, but the data suggest that on an evolutionary timescale octocorals can switch more easily between mixotrophy and heterotrophy compared to scleractinian corals, which coincides with a low reliance on photosynthetic carbon gain in the former group of organisms.

OBJECTIVE

Prostanoid EP(4) receptor antagonists may have therapeutic utility in the treatment of migraine since EP(4) receptors have been shown to be involved in prostaglandin (PG)E(2)-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP(4) receptor antagonist.

METHODS

BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP(4) receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo.

RESULTS

BGC20-1531 exhibited high affinity at recombinant human EP(4) receptors expressed in cell lines (pK(B) 7.6) and native EP(4) receptors in human cerebral and meningeal artery (pK(B) 7.6-7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE(2)-induced vasodilatation of human middle cerebral (pK(B) 7.8) and meningeal (pK(B) 7.6) arteries in vitro, but had no effect on responses induced by PGE(2) on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1-10 mg.kg(-1) i.v.) caused a dose-dependent antagonism of the PGE(2)-induced increase in canine carotid blood flow in vivo.

CONCLUSIONS

BGC20-1531 is a potent and selective antagonist at EP(4) receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache.

This study explores the organisation and neurochemical nature of the projections from the zona incerta (ZI) to the basal ganglia. Sprague-Dawley rats were anaesthetised with ketamine (100 mg/kg) and Rompun (10 mg/kg), and injections of cholera toxin subunit B were made into each of the following nuclei: the ZI, the substantia nigra (SN), the pedunculopontine tegmental nucleus (PpT), and the entopeduncular nucleus (Ep). Brains were aldehyde fixed, sectioned, and processed using standard methods. Tracer-labelled sections were then doubly labelled with antibodies to glutamate (Glu), nitric oxide synthase (NOS), parvalbumin (Pv), or glutamic acid decarboxylase (GAD; the latter two are markers for GABAergic cells); these neurochemicals characterise most types of ZI cells. After ZI injections, labelling was nonuniform across the different basal ganglia nuclei. The bulk of labelling, both anterograde and retrograde, was seen in the SN and PpT and, to a lesser extent, within the other nuclei of the basal ganglia (e.g., caudate-putamen, globus pallidus, subthalamus, Ep). In the SN, labelling was found in both major parts of the nucleus, the pars compacta and pars reticulata. Within the PpT, however, the bulk of labelling was limited to only one of the two sectors of the nucleus, namely, the pars dissipata (PpTd). The pars compacta of the PpT (PpTc) remained largely free of labelled profiles. After CTb injections into three basal ganglia nuclei (SN, PpT, Ep), most labelled cells in the ZI were glutamate+ and very few were NOS+ or gamma-aminobutyric acidergic. Overall, the results indicate that the ZI is in a position to influence preferentially the activity of the SN and PpTd of the basal ganglia via an excitatory, glutamatergic input.

OBJECTIVE

Retinoblastoma may exhibit variable hyperintensities on DWI, resulting in different values in the ADC maps, depending on their histology and cellularity. However, EP-based DWI has susceptibility artifacts and image distortions, which make DWI of the orbit a challenging technique. The aim of this study was to investigate the feasibility of single-shot turbo spin-echo (HASTE) DWI in the evaluation of children with retinoblastoma and to assess the value of ADC maps in differentiating viable and necrotic tumor tissue.

METHODS

Two radiologists assessed conventional MR images, DWI, and ADC maps of 17 patients with retinoblastoma (n = 17 eyes). Non-EP DWI was performed by using a HASTE sequence with b-values of 0 and 1000 s/mm(2). ADC values were measured for enhancing and nonenhancing tumor tissue. ADC maps were compared with histopathologic findings regarding tumor differentiation and viability.

RESULTS

On DWI, vital tumor tissue showed hyperintensity with negligible intensity of surrounding vitreous. The difference in mean (range) ADC values between enhancing (1.03 [0.72-1.22] × 10(-3) mm(2) s(-1)) and nonenhancing (1.47 [0.99-1.80] × 10(-3) mm(2) s(-1)) parts of retinoblastoma was statistically significant (P < .0005). Nonenhancing tumor parts showed a significantly lower ADC compared with vitreous (2.67 [2.24-3.20]×10(-3) mm(2) s(-1)) (P < .0005) and subretinal fluid (2.20 [1.76-2.96] × 10(-3) mm(2) s(-1)) (P < .0005). Histopathologically, low ADC values (enhancing tumor part) correlated to viable tumor tissue, whereas intermediate ADC values (nonenhancing tumor parts) correlated to necrotic tumor tissue.

CONCLUSIONS

HASTE DWI allowed adequate characterization of retinoblastoma, and ADC is a helpful tool to differentiate viable and necrotic tumor tissue and might be valuable in monitoring the response to eye-preserving therapies.

Intradermal injection of substance P elicits an itch sensation in human subjects and an itch-associated response in mice. The substance P-induced itch-associated response in mice is not inhibited by antihistamine. Therefore, the mechanisms of substance P-induced itch-associated response are unclear. In this study, we demonstrated one of the mechanisms. Substance P induces an arachidonate cascade to produce prostaglandins and leukotriene. In this study we considered whether arachidonate metabolites are involved in the substance P-induced itch-associated response. A phospholipase A(2) inhibitor arachidoryltrifluoromethyl ketone inhibited the substance P-induced itch-associated response in mice. Pre treatment with the glucocorticoids betamethasone and dexamethasone also produced inhibition of the substance P-induced itch-associated response in mice as well as humans. The 5-lipoxygenase inhibitor zileuton, but not the cyclooxygenase inhibitors indomethacin and diclofenac, suppressed substance P-induced itch-associated response. The leukotriene B(4) receptor antagonist 5-[2-(2-carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]valeric acid produced inhibition, whereas pranlukast (leukotriene C(4)/D(4)/E(4) receptor antagonist) and 5(Z)-7-[1S,2S, 3S,5R-3-(trans-b-styren)sulfonamido-6,6-dimethylbi cyclo(3,1,1)hept-2-yl]-5-heptenoic acid (EP(1) receptor antagonist) were without effect. Furthermore, when the production of leukotriene B(4) and prostaglandin E(2) was measured in skin injected with substance P and in mouse keratinocytes applied with substance P, the level of both products increased. As leukotriene B(4), but not prostaglandin E(2), also induces the itch-associated response in mice, these results suggest that leukotriene B(4) and keratinocytes, cutaneous cells which produced leukotriene B(4), play an important role in substance P-induced itch-scratch response in mice. Leukotriene B(4) receptor antagonist and 5-lipoxygenase inhibitor may be novel antipruritic drugs.

OBJECTIVE

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product.

METHODS

NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR.

RESULTS

Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05).

CONCLUSIONS

O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.