Following acceptance of clozapine as a superior antipsychotic agent with low risk of adverse extrapyramidal syndromes (EPS), such as dystonia, parkinsonism, akathisia or tardive dyskinesia, several novel antipsychotic drugs have been developed with properties modelled on those of clozapine. Though generally considered 'atypical' in their relatively low risk of inducing EPS, these agents vary considerably in their pharmacology and impact on neurological functioning. Although few comparative data are available, the atypical antipsychotics can be tentatively ranked by EPS risk (excluding akathisia and neuroleptic malignant syndrome) in the following order: clozapine < quetiapine < olanzapine = ziprasidone. At higher doses, risperidone is ranked with a higher EPS risk than olanzapine and ziprasidone, but its risk of EPS is lower with lower doses. In general, this ranking is inversely related to antidopaminergic (D2 receptor) potency. The high antiserotonergic (5-HT2A receptor) potency of risperidone, clozapine, ziprasidone and olanzapine, but not quetiapine, as well as the antimuscarinic activity of olanzapine and clozapine may also limit EPS. For the treatment of psychotic reactions to dopamine agonist therapy in Parkinson's disease, clozapine is both effective and relatively well tolerated; quetiapine may be tolerated, olanzapine is not well tolerated, risperidone is poorly tolerated, and amisulpride and ziprasidone have not been well evaluated. Clozapine, perhaps because of its anticholinergic activity, can reduce parkinsonian tremor. It is useful for ongoing psychosis with tardive dyskinesia, especially for dystonic features. No atypical antipsychotic is clearly effective for motor abnormalities in Huntington's disease or Tourette's syndrome, and the effect of these drugs on other neurological disorders have been well evaluated in only small numbers of patients. In summary, with the exception of clozapine, and perhaps quetiapine, atypical antipsychotics have brought only relative avoidance of EPS, strongly encouraging continued searches for novel antipsychotic agents.

The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with lower risks for esophageal, gastric and colon cancers as well as other solid tumors. The antitumor effect of NSAIDs is mediated through cyclooxygenase-2 (COX-2)-dependent and -independent regulation of oncogenic and tumor-suppressive pathways. Recent discoveries have shed new light on the regulation of COX-2 at the molecular level in these cancers. Moreover, prostaglandin E(2) (PGE(2)), a COX-2-derived eicosanoid, has been found to affect numerous tumorigenic processes. In this connection, PGE(2) activates multiple intracellular signaling pathways, including (1) transactivation of epidermal growth factor receptor (EGFR); (2) protein kinase C-dependent, EGFR-independent activation of extracellular signal-regulated kinase (ERK) and the transcription factors activator protein-1 and c-Myc; (3) G-protein-mediated activation of beta-catenin/TCF-dependent transcription. Activation of these signaling pathways by PGE(2) is mediated by EP receptors whose inhibitors suppress gastrointestinal carcinogenesis. Taken together, COX-2 expression is dysregulated in many types of cancer and COX-2-derived PGE(2) elicits multiple oncogenic signals to promote carcinogenesis. Targeting PGE(2) signaling by EP receptor antagonists holds promise for the development of targeted therapy for the treatment of cancer.

In the introductory section an overview is given of the strategies which have been proposed in the search for side-effect free antipsychotics. Special attention is paid to the role of predominant 5HT2 receptor blockade over D2 blockade. Whereas D2 receptor blockade seems to be essential for the treatment of positive symptoms of schizophrenia, it also underlies the induction of extrapyramidal side effects (EPS). Predominant 5HT2 receptor blockade may reduce the EPS liability and can ameliorate negative symptoms of schizophrenia. We further report a nearly complete list of neuroleptics that are on the European market and eight new antipsychotics that recently entered clinical trial, 5HT2 and D2 receptor binding affinity (Ki values) and the rank order in affinity for various neurotransmitter receptor subtypes are also discussed. For the eight new antipsychotics and for six reference compounds the complete receptor binding profile (including 33 radioligand receptor binding and neurotransmitter uptake models) is reported. Furthermore, for a series of 120 compounds the relative affinity for D2 receptors and D3 receptors (a recently cloned new dopamine receptor subtype) is compared. Finally, original findings are reported for the new antipsychotic risperidone and for haloperidol and clozapine on the in vivo occupation of neurotransmitter receptors in various brain areas after systemic treatment of rats or guinea pigs. The receptor occupation by the drugs was measured ex vivo by quantitative receptor autoradiography. The receptor occupancy was related to the motor activity effects of the test compounds (measurements were done in the same animals) and to the ability of the drugs to antagonize various 5HT2 and D2 receptor mediated effects. With risperidone a high degree of central 5HT2 receptor occupation was achieved before other neurotransmitter receptors became occupied. This probably co-underlies the beneficial clinical properties of the drug. Antagonism of the various D2 receptor-mediated effects was achieved at widely varying degrees of D2 receptor occupancy, from just about 10% to more than 70%. For therapeutic application it may be of prime importance to carefully titrate drug dosages. Antipsychotic effects may be achieved at a relatively low degree of D2 receptor occupancy at which motor disturbances are still minimal. With drugs such as risperidone that produce shallow log dose-effect curves, differentiation between the various D2 receptor mediated effects may be made more easily, allowing EPS-free maintenance therapy of schizophrenic patients.

Prostaglandin E2 (PGE2) is one of the most important biologically active prostanoids found throughout the gastrointestinal tract. Despite the fact that PGE2 regulates many physiological functions of the gut including mucosal protection, gastrointestinal secretion and motility, it is implicated in the pathophysiology of inflammatory bowel diseases (IBD) and colorectal neoplasia. The varied biological functions exerted by PGE2 are through the pharmacologically distinct, G-protein coupled plasma membrane receptors termed EP receptors. Disruptions of various prostanoid receptor genes have helped in unravelling the physiological functions of these receptors. To date, all four subtypes of EP receptors have been individually knocked out in mice and various phenotypes have been reported for each subtype. Similarly, in vitro and in vivo studies using EP receptor agonists and antagonists have helped in uncoupling the diverse functions of PGE2 signalling involving distinct EP receptors in the gut. In this review, we will summarize and conceptualize the salient features of EP receptor subtypes, their regional functions in the gut and how expressions of EP receptors are altered during disease states.

Claudins are cell adhesion molecules working at tight junctions (TJs) that are directly involved in compartmentalization in multicellular organisms. The cochlea includes a rather peculiar compartment filled with endolymph. This compartment is characterized by high K+ concentration (approximately 150 mM) and a positive endocochlear potential (approximately 90 mV; EP), both indispensable conditions for cochlear hair cells to transduce acoustic stimuli to electrical signals. These conditions are thought to be generated by the stria vascularis, which is adjacent to the endolymph compartment. The stria vascularis itself constitutes an isolated compartment delineated by two epithelial barriers, marginal and basal cell layers. Because TJs of basal cells are primarily composed of claudin-11, claudin-11-deficient (Cld11-/-) mice were generated with an expectation that the compartmentalization in stria vascularis in these mice would be affected. Auditory brainstem response measurements revealed that Cld11-/- mice suffered from deafness; although no obvious gross morphological malformations were detected in Cld11-/- cochlea, freeze-fracture replica electron microscopy showed that TJs disappeared from basal cells of the stria vascularis. In good agreement with this, tracer experiments showed that the basal cell barrier was destroyed without affecting the marginal cell barrier. Importantly, in the endolymph compartment of Cld11-/- cochlea, the K+ concentration was maintained around the normal level (approximately 150 mM), whereas the EP was suppressed down to approximately 30 mV. These findings indicated that the establishment of the stria vascularis compartment, especially the basal cell barrier, is indispensable for hearing ability through the generation/maintenance of EP but not of a high K+ concentration in the endolymph.

Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is coordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at < or =1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum. Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.

This paper reports and illustrates in figurine style results obtained by electrical stimulation of the cortex in 20 patients and by recording of cortical evoked potentials (EPs) in 13 of these patients, whose surgery required wide exposure of the Rolandic or paracentral regions of the cortex. This study is unique in that cutaneous receptive fields related to specific cortical sites were defined by mechanical stimulation, as is done in animals, in contrast to electrical stimulation of peripheral nerves at fixed sites, as in scalp EP recordings. Observations were made on pre- and postcentral gyri, on the second somatic sensory-motor area, on the supplementary motor area, and on the supplementary sensory area. In two patients with phantom limb pain, the pain was elicited in one on stimulation of the postcentral arm area, and in the other on stimulation of the supplementary sensory leg area. Surgical removal of these areas had the immediate effect of abolishing the phantoms and the pain. Long-term follow-up review was not possible. In one patient with severe Parkinson's disease, stimulating currents subthreshold for the elicitation of movement resulted in disappearance of tremor and rigidity for short periods after stimulation of the precentral gyrus. The possible patterns of organization of the human pre- and postcentral areas are considered and compared with those of the chimpanzee and other primates. In patients in whom data from pre- and postcentral gyri were adequate, it appeared that the precentral face-arm boundary is situated 1 to 2 cm higher than the corresponding postcentral boundary.

Increased expression of COX-2 or VEGF-C has been correlated with progressive disease in certain cancers. Present study utilized several human breast cancer cell lines (MCF-7, T-47D, Hs578T and MDA-MB-231, varying in COX-2 expression) as well as 10 human breast cancer specimens to examine the roles of COX-2 and prostaglandin E (<em>EP</em>) receptors in VEGF-C expression or secretion, and the relationship of COX-2 or VEGF-C expression to lymphangiogenesis. We found a strong correlation between COX-2 mRNA expression and VEGF-C expression or secretion levels in breast cancer cell lines and VEGF-C expression in breast cancer tissues. Expression of LYVE-1, a selective marker for lymphatic endothelium, was also positively correlated with COX-2 or VEGF-C expression in breast cancer tissues. Inhibition of VEGF-C expression and secretion in the presence of COX-1/2 or COX-2 inhibitors or following downregulation of COX-2 with COX-2 siRNA established a stimulatory role COX-2 in VEGF-C synthesis by breast cancer cells. <em>EP</em>1 as well as <em>EP</em>4 receptor antagonists inhibited VEGF-C production indicating the roles of <em>EP</em>1 and <em>EP</em>4 in VEGF-C upregulation by endogenous PGE2. Finally, VEGF-C secretion by MDA-MB-231 cells was inhibited in the presence of kinase inhibitors for Her-2/neu, Src and p38 MAPK, indicating a requirement of these kinases for VEGF-C synthesis. These results, for the first time, demonstrate a regulatory role of COX-2 in VEGF-C synthesis (and thereby lymphangiogenesis) in human breast cancer, which is mediated at least in part by <em>EP</em>1/<em>EP</em>4 receptors.

BACKGROUND

The advent of fibrin-binding molecular magnetic resonance (MR) contrast agents and advances in coronary MRI techniques offers the potential for direct imaging of coronary thrombosis. We tested the feasibility of this approach using a gadolinium (Gd)-based fibrin-binding contrast agent, EP-2104R (EPIX Medical Inc), in a swine model of coronary thrombus and in-stent thrombosis.

RESULTS

Ex vivo and in vivo sensitivity of coronary MR thrombus imaging was tested by use of intracoronarily delivered Gd-DTPA-labeled fibrinogen thrombi (n=6). After successful demonstration, in-stent coronary thrombosis was induced by x-ray-guided placement of thrombogenic-coated, MR-lucent stents (n=5). After stent placement, 60 micromol of EP-2104R was injected via the left main coronary artery. Free-breathing, navigator-gated 3D coronary MR angiography and thrombus imaging were performed (1) before and after stent placement and (2) before and after EP-2104R. Thrombi were confirmed by x-ray angiography and autopsy. Fibrinogen thrombi: 5 of 6 intracoronarily delivered Gd-labeled fibrinogen clots (approximately 250 micromol/L Gd) were visible on MRI and subsequently confirmed by x-ray angiography. In-stent thrombi: in-stent thrombosis was observed in all stents after EP-2104R. Four of 5 thrombi were confirmed by x-ray angiography. Chemical analysis of 2 thrombi demonstrated 99 to 147 micromol/L Gd.

CONCLUSIONS

We demonstrate the feasibility of MRI of coronary thrombus and in-stent thrombosis using a novel fibrin-binding molecular MR contrast agent. Potential applications include detection of coronary in-stent thrombosis or thrombus burden in patients with acute coronary syndromes.

Renal cyclooxygenase 1 and 2 activity produces five primary prostanoids: prostaglandin E2, prostaglandin F2alpha, prostaglandin I2, thromboxane A2, and prostaglandin D2. These lipid mediators interact with a family of distinct G protein-coupled prostanoid receptors designated <em>EP</em>, FP, IP, TP, and DP, respectively, which exert important regulatory effects on renal function. The intrarenal distribution of these prostanoid receptors has been mapped, and the consequences of their activation have been partially characterized. FP, TP, and <em>EP</em>1 receptors preferentially couple to an increase in cell calcium. <em>EP</em>2, <em>EP</em>4, DP, and IP receptors stimulate cyclic AMP, whereas the <em>EP</em>3 receptor preferentially couples to Gi, inhibiting cyclic AMP generation. <em>EP</em>1 and <em>EP</em>3 mRNA expression predominates in the collecting duct and thick limb, respectively, where their stimulation reduces NaCl and water absorption, promoting natriuresis and diuresis. The FP receptor is highly expressed in the distal convoluted tubule, where it may have a distinct effect on renal salt transport. Although only low levels of <em>EP</em>2 receptor mRNA are detected in the kidney and its precise intrarenal localization is uncertain, mice with targeted disruption of the <em>EP</em>2 receptor exhibit salt-sensitive hypertension, suggesting that this receptor may also play an important role in salt excretion. In contrast, <em>EP</em>4 receptor mRNA is predominantly expressed in the glomerulus, where it may contribute to the regulation of glomerular hemodynamics and renin release. The IP receptor mRNA is highly expressed near the glomerulus, in the afferent arteriole, where it may also dilate renal arterioles and stimulate renin release. Conversely, TP receptors in the glomerulus may counteract the effects of these dilator prostanoids and increase glomerular resistance. At present there is little evidence for DP receptor expression in the kidney. These receptors act in a concerted fashion as physiological buffers, protecting the kidney from excessive functional changes during periods of physiological stress. Nonsteroidal anti-inflammatory drug (NSAID)-mediated cyclooxygenase inhibition results in the loss of these combined effects, which contributes to their renal effects. Selective prostanoid receptor antagonists may provide new therapeutic approaches for specific disease states.

RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMbeta. After transfection at approximately 5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced con canavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET- procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.

Mutations or multiplications in alpha-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type alpha-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating alpha-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of alpha-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human alpha-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed alpha-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in alpha-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing alpha-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. alpha-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of alpha-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated alpha-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and alpha-synuclein-induced neurotoxicity in synucleinopathy.

OBJECTIVE

Currently available clinical and molecular factors provide still an insufficient prognostic and predictive assessment for patients with epithelial ovarian cancer (EOC). To identify a potential molecular target and prognostic/predictive factor for EOC, we investigated in a retrospective study the prognostic value of Ep-CAM overexpression in EOC.

METHODS

We assessed by immunohistochemistry the expression of the Ep-CAM antigen on tissue microarrays containing paraffin-embedded tissue samples of 199 patients with documented EOC. Patients were operated for ovarian cancer in the period between June 1980 and January 2000.

RESULTS

We observed a rate of Ep-CAM overexpression of 68.8%. Ep-CAM overexpression was significantly related to a decreased overall survival (P = 0.036). The prognostic power of Ep-CAM overexpression was particularly strong in patients with stage III and IV disease. In fact, in this subgroup, median overall survival was twofold higher in patients without as compared to patients with Ep-CAM overexpression (46 vs. 23 months, P < 0.01). Univariate analysis revealed a correlation with histologic grade. We observed a significantly higher rate of Ep-CAM overexpression (83.5%) in grade 3 tumors. Histologic subtypes associated with a higher rate of Ep-CAM overexpression were serous carcinoma, squamous cell carcinoma, undifferentiated carcinoma, clear cell carcinoma, and endometrioid carcinoma. Cox regression analysis showed Ep-CAM overexpression to be an independent prognostic marker (P = 0.037, RR = 1.64).

CONCLUSIONS

This retrospective analysis demonstrates for the first time an independent prognostic value of Ep-CAM overexpression in patients with EOC. Ovarian cancer patients with Ep-CAM overexpressing tumors are frequent and would qualify for treatment with Ep-CAM-specific immunotherapeutic approaches.

The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.

Evoked potentials (EPs) were recorded from 991 frontal and peri-rolandic sites (106 electrodes) in 36 patients during an auditory discrimination task with target and non-target (distractor) rare stimuli. Variants of this task explored the effects of attention, dishabituation and stimulus characteristics (including modality). Rare stimuli evoked a widespread triphasic waveform with negative, positive and negative peaks at about 210, 280 and 390 msec, respectively. This waveform was identified with the scalp EP complex termed the N2a/P3a/slow wave and associated with orienting. It was evoked by rare target and distractor auditory and visual stimuli, as well as by rare stimulus repetitions or omissions. Across most frontal trajectories, N2a/P3a/SW amplitudes changed only slowly with distance. However, large (120 microV) P3as with steep voltage gradients were observed laterally, especially near the inferior frontal sulcus, and clear inversions of the P3a were noted in the orbito-frontal and the anterior cingulate cortices. The frontal P3a was earlier to distractor than to target stimuli, but only in some sites and with a latency difference much smaller than that observed at the scalp. Frontal P3a latencies were significantly shorter than those recorded simultaneously at the scalp and often were also shorter than P3a latency in the parietal or temporal lobes. In summary, this study demonstrates an early P3a-like activity that polarity inverts over short distances in the medial frontal lobe, and that it has a significantly shorter latency than similar potentials recorded in the temporal and parietal cortices.

Our understanding of the key players involved in the differential regulation of T-cell responses during inflammation, infection and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. With respect to this, the inhibitory role of the lipid mediator prostaglandin E(2) (PGE(2)) in T-cell immunity has been documented since the 1970s. Studies that ensued investigating the underlying mechanisms substantiated the suppressive function of micromolar concentrations of PGE(2) in T-cell activation, proliferation, differentiation and migration. However, the past decade has seen a revolution in this perspective, since nanomolar concentrations of PGE(2) have been shown to potentiate Th1 and Th17 responses and aid in T-cell proliferation. The understanding of concentration-specific effects of PGE(2) in other cell types, the development of mice deficient in each subtype of the PGE(2) receptors (EP receptors) and the delineation of signalling pathways mediated by the EP receptors have enhanced our understanding of PGE(2) as an immune-stimulator. PGE(2) regulates a multitude of functions in T-cell activation and differentiation and these effects vary depending on the micro-environment of the cell, maturation and activation state of the cell, type of EP receptor involved, local concentration of PGE(2) and whether it is a homeostatic or inflammatory scenario. In this review, we compartmentalize the various aspects of this complex relationship of PGE(2) with T lymphocytes. Given the importance of this molecule in T-cell activation, we also address the possibility of using EP receptor antagonism as a potential therapeutic approach for some immune disorders.

In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express 'cooperative traits', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.

A range of prostanoid agonists were tested for activity on isolated ring preparations of piglet saphenous vein. The selective TxA2-mimetic (TP-receptor agonist), U-46619, contracted the preparation in a concentration-related fashion. These contractions were inhibited by the TP-receptor blocking drug, GR32191B, producing a pA2 of 7.8 (slope = 1.6). Prostanoid-induced relaxant responses were studied on preparations which had been pre-contracted using an EC60 concentration of phenylephrine (mean EC60 = 0.97 microM), in the presence of GR32191B (1 microM), to block contractile TP-receptors. Under these conditions, PGD2, PGE2, PGF2 alpha, PGI2, and U-46619, all caused concentration-related relaxation. PGE2 was the most potent agonist (EC50 = 0.23nM), whereas, all of the other agonists were at least 1,000-fold weaker, providing strong evidence for the presence of inhibitory <em>EP</em>-receptors. The selective synthetic <em>EP</em>-agonists, sulprostone (<em>EP</em>1/<em>EP</em>3) and AH13205X (<em>EP</em>2), were next tested for relaxant activity. While both compounds caused concentration-related relaxant activity, they were respectively 6,000 and 11,000-fold less potent than PGE2. The potent TP-receptor blocking drugs, AH22921X and AH23848B, were both weak antagonists of PGE2 but not isoproterenol-induced relaxant responses of piglet saphenous vein in a concentration-related fashion. These two compounds had pA2 values against PGE2 of 5.3 and 5.4 respectively, with regression slopes not significantly different from unity. In contrast, neither compound at a concentration of 30 microM had any antagonist activity against prostanoid-induced effects on guinea-pig fundus (<em>EP</em>1), rabbit ear artery (<em>EP</em>2) or guinea-pig vas deferens (<em>EP</em>3). In conclusion, the piglet saphenous vein contains TP-receptors mediating smooth muscle contraction, and a PGE2-specific (<em>EP</em>) receptor mediating relaxation. The inhibitory <em>EP</em>-receptor does not appear to be of the <em>EP</em>1, <em>EP</em>2 or <em>EP</em>3-subtypes, and appears therefore to be a novel subtype which we tentatively term <em>EP</em>4, and the potent TP-receptor blocking drugs, AH22921X and AH23848B, appear to be weak, but specific <em>EP</em>4-receptor blocking drugs.

Elucidation of the mechanism(s) by which dietary fish oil, enriched in eicosapentaenoic acid (EPA, 20:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-3)], suppresses the inflammatory process is essential in maximizing this potentially therapeutic effect. Murine T-lymphocyte function and signal transduction were examined in response to a low fat, short term diet enriched in highly purified EPA or DHA ethyl esters. For 10 d, mice were fed comparable diets containing either 3% safflower oil ethyl esters (SAF), 2% SAF + 1% arachidonic acid triglyceride (AA), 2% SAF + 1% EPA, or 2% SAF + 1% DHA. Concanavalin A-induced T-lymphocyte proliferation in splenocyte cultures was significantly suppressed by dietary EPA and DHA while AA had no effect relative to the SAF control. The suppressed proliferative response in EPA- and DHA-fed mice was preceded temporally by a significant reduction in IL-2 secretion. Kinetics of mitogen-induced diacyl-sn-glycerol (DAG) and ceramide production did not differ significantly between SAF and AA diet groups. In contrast, DAG production was significantly suppressed in EP- and DHA-fed mice relative to the SAF and AA groups. The reduced DAG mass was paralleled by reduced ceramide mass following EPA and DHA feeding compared to the SAF and AA groups. Thus, low dose, short term dietary exposure to highly purified EPA or DHA appears to suppress mitogen-induced T-lymphocyte proliferation by inhibiting IL-2 secretion, and these events are accompanied by reductions in the production of essential lipid second messengers, DAG and ceramide.

In many bacterial pathogens, the second messenger c-di-GMP stimulates the production of an exopolysaccharide (EPS) matrix to shield bacteria from assaults of the immune system. How c-di-GMP induces EPS biogenesis is largely unknown. Here, we show that c-di-GMP allosterically activates the synthesis of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a major extracellular matrix component of Escherichia coli biofilms. C-di-GMP binds directly to both PgaC and PgaD, the two inner membrane components of the poly-GlcNAc synthesis machinery to stimulate their glycosyltransferase activity. We demonstrate that the PgaCD machinery is a novel type c-di-GMP receptor, where ligand binding to two proteins stabilizes their interaction and promotes enzyme activity. This is the first example of a c-di-GMP-mediated process that relies on protein-protein interaction. At low c-di-GMP concentrations, PgaD fails to interact with PgaC and is rapidly degraded. Thus, when cells experience a c-di-GMP trough, PgaD turnover facilitates the irreversible inactivation of the Pga machinery, thereby temporarily uncoupling it from c-di-GMP signalling. These data uncover a mechanism of c-di-GMP-mediated EPS control and provide a frame for c-di-GMP signalling specificity in pathogenic bacteria.

The role of Eps15 in clathrin-mediated endocytosis is supported by two observations. First, it interacts specifically and constitutively with the plasma membrane adaptor AP-2. Second, its NH2 terminus shows significant homology to the NH2 terminus of yeast End3p, necessary for endocytosis of alpha-factor. To gain further insight into the role of Eps15-AP-2 association, we have now delineated their sites of interactions. AP-2 binds to a domain of 72 amino acids (767-739) present in the COOH terminus of Eps15. This domain contains 4 of the 15 DPF repeats characteristic of the COOH-terminal domain of Eps15 and shares no homology with known proteins, including the related Epsl5r protein. Precipitation of proteolytic fragments of AP-2 with Eps15-derived fusion proteins containing the binding site for AP-2 showed that Eps15 binds specifically to a 40-kDa fragment corresponding to the ear of alpha-adaptin, a result confirmed by precipitation of Eps15 by alpha-adaptin-derived fusion proteins. Our data indicate that this specific part of AP-2 binds to a cellular component and provide the tools for investigating the functions of the association between AP-2 and Eps15.

A new technique for the detection of antibodies bound to proteins blotted onto nitrocellulose paper was developed. The method is rapid, sensitive, and does not require radioactive probes. Proteins transferred to nitrocellulose paper are first reacted with primary antibody followed by reaction with an alkaline phosphatase conjugated second antibody. The phosphatase activity is then visualized using an agar gel impregnated with the histochemical phosphatase stain 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (J. P. Horwitz, J. Chua, M. Noel, J. T. Donatti, and J. Freisler (1966) J. Med. Chem. 9, 447; Sigma Chemical Co., Technical bulletin No. 710-EP (1978]. Antigen-antibody complexes give rise to sharp, permanent blue stained bands both on the nitrocellulose paper and in the agar overlay gel. This procedure allows detection of bands containing less than 20 ng of protein.

An increased migratory phenotype exists in lung fibroblasts derived from patients with fibroproliferative lung disease. Prostaglandin E(2) (PGE(2)) suppresses fibroblast migration, but the receptor(s) and mechanism(s) mediating this action are unknown. Our data confirm that treatment of human lung fibroblasts with PGE(2) inhibits migration. Similar effects of butaprost, an E-prostanoid (<em>EP</em>) 2 receptor-specific ligand, implicate the <em>EP</em>2 receptor in migration-inhibitory signaling. Further, migration in fibroblasts deficient for the <em>EP</em>2 receptor cannot be inhibited by PGE(2) or butaprost, confirming the central role of <em>EP</em>2 in mediating these effects. Our previous data suggested that phosphatase and tensin homolog on chromosome ten (PTEN), a phosphatase that opposes the actions of phosphatidylinositol-3-kinase (PI3K), may be important in regulating lung fibroblast motility. We now report that both PGE(2) and butaprost increase PTEN phosphatase activity, without a concomitant increase in PTEN protein levels. This contributes to <em>EP</em>2-mediated migration inhibition, because migration in PTEN-null fibroblasts is similarly unaffected by <em>EP</em>2 receptor signaling. Increased PTEN activity in response to <em>EP</em>2 stimulation is associated with decreased tyrosine phosphorylation on PTEN, a mechanism known to regulate enzyme activity. Collectively, these data describe the novel mechanistic finding that PGE(2), via the <em>EP</em>2 receptor, decreases tyrosine phosphorylation on PTEN, resulting in increased PTEN enzyme activity and inhibition of fibroblast migration.

The pain field has been advocating for some time for the importance of teaching people how to live well with pain. Perhaps some, and maybe even for many, we might again consider the possibility that we can help people live well without pain. Explaining Pain (EP) refers to a range of educational interventions that aim to change one's understanding of the biological processes that are thought to underpin pain as a mechanism to reduce pain itself. It draws on educational psychology, in particular conceptual change strategies, to help patients understand current thought in pain biology. The core objective of the EP approach to treatment is to shift one's conceptualization of pain from that of a marker of tissue damage or disease to that of a marker of the perceived need to protect body tissue. Here, we describe the historical context and beginnings of EP, suggesting that it is a pragmatic application of the biopsychosocial model of pain, but differentiating it from cognitive behavioral therapy and educational components of early multidisciplinary pain management programs. We attempt to address common misconceptions of EP that have emerged over the last 15 years, highlighting that EP is not behavioral or cognitive advice, nor does it deny the potential contribution of peripheral nociceptive signals to pain. We contend that EP is grounded in strong theoretical frameworks, that its targeted effects are biologically plausible, and that available behavioral evidence is supportive. We update available meta-analyses with results of a systematic review of recent contributions to the field and propose future directions by which we might enhance the effects of EP as part of multimodal pain rehabilitation. Perspective: EP is a range of educational interventions. EP is grounded in conceptual change and instructional design theory. It increases knowledge of pain-related biology, decreases catastrophizing, and imparts short-term reductions in pain and disability. It presents the biological information that justifies a biopsychosocial approach to rehabilitation.