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Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).

AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg(2+). Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.

Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA-auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. (1) H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K(M) values of 43 and 270 μM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg(2+) , Mn(2+) and Co(2+) , although no typical (N/D)DXXD binding motif was found in the sequence., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

E-health and systems related to the electronic patient record (EPR) are seen as important factors in the development of the health care System. In 2004 European Commission had adopted e-Health Action Plan, which indicated the development directions of European e-Health. In Poland, the main development trends and Government course of actions in this regard, are contained in the document Computerization plan "e-Health Poland" 2009-2015. The European Commission defines e-Health as an application of tools and services, information and communication technologies in healthcare. EPR is a collection of patient data that are stored in a certain place and it is possible to access them. E-health and EPR are closely related to the concept of interoperability. Denmark is one of the countries in which the information services and information technology in healthcare is mostly used. The introduction of ERP involves a lot of positive effects. Using the ERP, stored data can be optimally used by both physicians and patients. However, also risks associated with data security need to be considered. Furthermore, the Polish law defines in great detail the issues associated with creating, storing and sharing medical records (1). According to the Act from 17 February 2005, concerning the computerization of public service activities, it possible to keep medical documentation in electronically form.

One of the solutions aimed at improving the functioning of the healthcare system in Poland is to introduce patients' co-payment for public healthcare services. In all countries where the healthcare system is at a high level there already exists a co-payment system and it is regarded by many specialists as a necessary and indispensable condition for the proper functioning of healthcare. The aim of this study was to show respondents' attitudes and opinions regarding the proposal of introduction co-payments as and additional form of financing medical care. The questionnaire survey covered a group of 2,409 persons (50.7% men and 49.3% women). Most respondents, despite the overall rising dissatisfaction with the quality and availability of medical services do not see the need for co-payments. The opinion about the implementation of co-payments. The opinion about the implementation of co-payments depends on many factors, to the most important belong age, education, place of residence and income. More often, the co-payments is in favour of young people in good health condition, who live in big cities, having a university degree and determining their financial situation as good. Before the introduction of co-payment - certain social groups, which would be exempt from additional fees, should be specified. To the highest costs that patients are able to carry belong: paying for a home visit of family doctor or specialist, for surgical procedures, and for complex tests performed during the hospital stay (including computed tomography, magnetic resonance imaging).

One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.

The aim of the financial and economic analysis, conducted in March 2010, was to identify all significant factors that had a positive influence on the restructuring process in the health service centre (Samodzielny Zespół Publicznych Zakładów Lecznictwa Otwartego Warszawa--Mokotów) in Warsaw. Within the framework of the analysis, financial data form time period 1999-2009 were analyzed. Also the managing director and financial director were interviewed. Taking into consideration research results it can be stated that not a single factor but a collection of the purposeful efforts influenced the improvement of the health service centre condition. Apart from received public help, the most significant factors include: rational restructuring process, managing of personnel development, professionally managed financial department, cooperation between departments, good internal communication and use of modern management techniques.

Two dimethylallyltryptophan synthases, FgaPT2 and 7-DMATS, which catalysed the prenylation of L-tryptophan at positions C4 and C7, respectively, have been recently identified in Aspergillus fumigatus and proven biochemically. These enzymes were successfully used for the production of monoprenylated indole derivatives. In this study, we showed that C4,C7-diprenylated indole derivatives, e.g. 4,7-di-(dimethylallyl)-L-tryptophan, 4,7-di-(dimethylallyl)-L-abrine and 4,7-di-(dimethylallyl)-11-methyltryptophan, could be conveniently produced by tandem incubation of both enzymes. The structures of the isolated enzymatic products were elucidated by NMR and MS analyses. High conversion yields of up to 93% were achieved by an incubation sequence of FgaPT2 followed by 7-DMATS. The results reported in this study demonstrated the potential of secondary metabolite enzymes as promising tools for the production of designed compounds.

Ergot alkaloids are toxins and important pharmaceuticals that are produced biotechnologically on an industrial scale. The first committed step of ergot alkaloid biosynthesis is catalyzed by dimethylallyl tryptophan synthase (DMATS; EC 2.5.1.34). Orthologs of DMATS are found in many fungal genomes. We report here the x-ray structure of DMATS, determined at a resolution of 1.76 A. A complex of DMATS from Aspergillus fumigatus with its aromatic substrate L-tryptophan and with an analogue of its isoprenoid substrate dimethylallyl diphosphate reveals the structural basis of this enzyme-catalyzed Friedel-Crafts reaction, which shows strict regiospecificity for position 4 of the indole nucleus of tryptophan as well as unusual independence of the presence of Mg(2+) ions. The 3D structure of DMATS belongs to a rare beta/alpha barrel fold, called prenyltransferase barrel, that was recently discovered in a small group of bacterial enzymes with no sequence similarity to DMATS. These bacterial enzymes catalyze the prenylation of aromatic substrates in the biosynthesis of secondary metabolites (i.e., a reaction similar to that of DMATS).

Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.

Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain sigma70-dependent promoters. A number of promoters that are dependent on alternative sigma-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived sigma54-dependent Po promoter and several other sigma54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro sigma54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the beta- and beta'-subunits of core RNA polymerase that allow high levels of transcription from the sigma54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with sigma-factors to favor sigma54-holoenzyme formation over that with sigma70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent sigma70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of sigma54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent sigma70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative sigma-factors is controlled to meet cellular requirements.

Cbl is a member of the large family of LysR-type transcriptional regulators (LTTRs) common in bacteria and found also in Archaea and algal chloroplasts. The function of Cbl is required in Escherichia coli for expression of sulphate starvation-inducible (ssi) genes, associated with the biosynthesis of cysteine from organic sulphur sources (sulphonates). Here, we report the crystal structure of the cofactor-binding domain of Cbl (c-Cbl) from E. coli. The overall fold of c-Cbl is very similar to the regulatory domain (RD) of another LysR family member, CysB. The RD is composed of two subdomains enclosing a cavity, which is expected to bind effector molecules. We have constructed and analysed several full-length Cbl variants bearing single residue substitutions in the RD that affect cofactor responses. Using in vivo and in vitro transcription assays, we demonstrate that pssuE, a Cbl responsive promoter, is down-regulated not only by the cofactor, adenosine phosphosulphate (APS), but also by thiosulphate, and, that the same RD determinants are important for the response to both cofactors. We also demonstrate the effects of selected site-directed mutations on Cbl oligomerization and discuss these in the context of the structure. Based on the crystal structure and molecular modelling, we propose a model for the interaction of Cbl with adenosine phosphosulphate.

Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Abeta42 peptide from the beta-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Abeta42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Abeta42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Abeta40, Abeta42, and sAPPbeta, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Abeta secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Abeta42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

RNA interference (RNAi) high-throughput screening (HTS) experiments carried out using large (>5000 short interfering [si]RNA) libraries generate a huge amount of data. In order to use these data to identify the most effective siRNAs tested, it is critical to adopt and develop appropriate statistical methods. To address the questions in hit selection of RNAi HTS, we proposed a quartile-based method which is robust to outliers, true hits and nonsymmetrical data. We compared it with the more traditional tests, mean +/- k standard deviation (SD) and median +/- 3 median of absolute deviation (MAD). The results suggested that the quartile-based method selected more hits than mean +/- k SD under the same preset error rate. The number of hits selected by median +/- k MAD was close to that by the quartile-based method. Further analysis suggested that the quartile-based method had the greatest power in detecting true hits, especially weak or moderate true hits. Our investigation also suggested that platewise analysis (determining effective siRNAs on a plate-by-plate basis) can adjust for systematic errors in different plates, while an experimentwise analysis, in which effective siRNAs are identified in an analysis of the entire experiment, cannot. However, experimentwise analysis may detect a cluster of true positive hits placed together in one or several plates, while platewise analysis may not. To display hit selection results, we designed a specific figure called a plate-well series plot. We thus suggest the following strategy for hit selection in RNAi HTS experiments. First, choose the quartile-based method, or median +/- k MAD, for identifying effective siRNAs. Second, perform the chosen method experimentwise on transformed/normalized data, such as percentage inhibition, to check the possibility of hit clusters. If a cluster of selected hits are observed, repeat the analysis based on untransformed data to determine whether the cluster is due to an artifact in the data. If no clusters of hits are observed, select hits by performing platewise analysis on transformed data. Third, adopt the plate-well series plot to visualize both the data and the hit selection results, as well as to check for artifacts.

This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.

Cbl (CysB-like protein) is a member of the family of LysR-type transcriptional regulators (LTTRs) and controls genes engaged in sulfur assimilation in Escherichia coli. It has been postulated that adenosine 5-phosphosulfate (APS) is responsible for abolishing Cbl-activated transcription from the ssu promoter (Bykowski et al., 2002). To elucidate the structural basis of Cbl function and to confirm the role of APS as an anti-inducer, the cofactor-binding domain of Cbl (c-Cbl, MW = 26 kDa) was cloned, purified and crystallized in the presence of APS. The crystals belong to space group C222(1), but show substantial variation of the unit-cell parameters and diffraction anisotropy. Despite this, X-ray data extending to 3.0 A resolution have been collected and solution of the structure by molecular replacement is in progress.

Many libraries are grappling with the new freedom to link to the Internet directly from a Web-based catalog. A conservative approach that locates only Web sites replicating a library's print holdings is described. Methods for locating URLs include reviewing front matter in reference materials, scouring the Internet for material produced both in print and full text on the Web, electronic mailing lists, publishers' promotional materials, conferences, and journal articles. Readers are encouraged to make links from the catalog to any original material produced in Web-readable form by the university, including dissertations and conference proceedings.

The relationship between local rates of cerebral glucose utilization (ICMRglc) and glucose transporter expression was examined during physiologic activation of the hypothalamoneurohypophysial system. Three days of water deprivation, which is known to activate the hypothalamoneurohypophysial system, resulted in increased ICMRglc and increased concentrations of GLUT1 and GLUT3 in the neurohypophysis; mRNA levels of GLUT1 and GLUT3 were decreased and increased, respectively. Water deprivation also increased ICMRglc in the hypothalamic supraoptic and paraventricular nuclei; mRNA levels of GLUT1 and GLUT3 appeared to increase in these nuclei, but the changes did not achieve statistical significance. Restoration of water for 3 to 7 days reversed all observed changes in GLUT expression (protein and mRNA): restoration of water also reversed changes in ICMRglc in both the neurohypophysis and the hypothalamic nuclei. These results indicate that under conditions of neural activation and recovery, changes in ICMRglc and the levels of GLUT1 and GLUT3 are temporally correlated in the neurohypophysis and raise the possibility that GLUT1 and GLUT3 transporter expression may be regulated by chronic changes in functional activity. In addition, increases in the expression of GLUT5 mRNA in the neurohypophysis after dehydration provide evidence for involvement of microglial activation.

Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [3H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC50s of 0.2-2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all 10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms.

The transport of glucose across the blood-brain barrier (BBB) is mediated by the high molecular mass (55-kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2-N-[4-(1 -azi-2,2,2-trifluoroethyl)[2-3H]propyl]-1,3-bis(D-mannose-4-ylo xy)-2-propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous-release insulin pellets (6 U/day) for a 12- to 14-day duration; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14- to 21-day duration. Hypoglycemia resulted in 25-45% increases in regional BBB permeability-surface area (PA) values for D-[14C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarly, there was a 23+/-4% increase in total GLUT1/mg of microvessel protein and a 52+/-13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55-kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30-40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.

The effect of continuous intracerebroventricular (i.c.v.) infusion of oxytocin (OT) on the release of OT and vasopressin (VP) following osmotic stimulation was studied in ovariectomized rats treated peripherally with gonadal steroids to simulate late gestation/lactation. Artificial cerebrospinal fluid (CSF) with or without OT (2 ng/microg) was infused (0.5 microl/h) i.c.v. continuously for 7 days along with sequential peripheral administration of progesterone (2 mg/kg i.m.) for 4 days, then 17-beta-estradiol (200 microg/kg i.m.) for 2 days. Following 7 days of OT infusion, isotonic (0.15 mol/l NaCl) or hypertonic (1.5 mol/l NaCl) saline was injected (15 ml/kg s.c.); the animals were decapitated 1 h later. Animals infused centrally with OT had higher basal levels of OT in plasma (p < 0.01 vs. CSF). While osmotic stimulation increased plasma levels of both OT and VP (0.15 mol/l NaCl < 1.5 mol/l NaCl; p < 0.01), only circulating VP was enhanced further (p < 0.01) in animals infused with OT compared with those receiving CSF. These changes in hormone levels could not be explained by differences in neural lobe stores of OT or VP or by alterations in daily water intake during the infusion period. Thus, chronic i.c.v. infusion of OT stimulates basal release of OT and increases the response of the VP system to osmotic stimulation.

Glucose is the principle energy source for mammalian brain. Delivery of glucose from the blood to the brain requires its transport across the endothelial cells of the blood-brain barrier and across the plasma membranes of neurons and glia, which is mediated by the facilitative glucose transporter proteins. The two primary glucose transporter isoforms which function in cerebral glucose metabolism are GLUT1 and GLUT3. GLUT1 is the primary transporter in the blood-brain barrier, choroid plexus, ependyma, and glia; GLUT3 is the neuronal glucose transporter. The levels of expression of both transporters are regulated in concert with metabolic demand and regional rates of cerebral glucose utilization. We present several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. These include normal cerebral development in the rat, Alzheimer's disease, neuronal differentiation in vitro, and dehydration in the rat.

Platelets derive most of their energy from anaerobic glycolysis; during activation this requirement rises approx. 3-fold. To accommodate the high glucose flux, platelets express extremely high concentrations (155+/-18 pmol/mg of membrane protein) of the most active glucose transporter isoform, GLUT3. Thrombin, a potent platelet activator, was found to stimulate 2-deoxyglucose transport activity 3-5-fold within 10 min at 25 degrees C, with a half-time of 1-2 min. To determine the mechanism underlying the increase in glucose transport activity, an impermeant photolabel, [2-3H]2N-4-(1-azi-2,2,2-trifluoethyl)benzoyl-1,3, -bis-(d-mannose-4-ylozy)-2-propylamine, was used to covalently bind glucose transporters accessible to the extracellular milieu. In response to thrombin, the level of transporter labelling increased 2.7-fold with a half-time of 1-2 min. This suggests a translocation of GLUT3 transporters from an intracellular site to the plasma membrane in a manner analogous to that seen for the translocation of GLUT4 in insulin-stimulated rat adipose cells. To investigate whether a similar signalling pathway was involved in both systems, platelets and adipose cells were exposed to staurosporin and wortmannin, two inhibitors of GLUT4 translocation in adipose cells. Thrombin stimulation of glucose transport activity in platelets was more sensitive to staurosporin inhibition than was insulin-stimulated transport activity in adipose cells, but it was totally insensitive to wortmannin. This indicates that the GLUT3 translocation in platelets is mediated by a protein kinase C not by a phosphatidylinositol 3-kinase mechanism. In support of this contention, the phorbol ester PMA, which specifically activates protein kinase C, fully stimulated glucose transport activity in platelets and was equally sensitive to inhibition by staurosporin. This study provides a cellular mechanism by which platelets enhance their capacity to import glucose to fulfil the increased energy demands associated with activation.

The effect of 101 Escherichia coli strains on growth of 90 Bacillus cereus strains on solid media was investigated. Only 9 E. coli strains (in particular the colicin +-generating ones) were antagonistic towards B. cereus, giving distinct growth-inhibition zones around the colonies.

The study was done on 60 strains of Bacillus cereus isolated from cases of food poisoning and from food products tested in current supervision. The highest sensitivity was found to the antibiotics: streptomycin, neomycin, gentamicin, amikacin, oxytetracycline, and sulphonamides: negram and sulfathiazole . All strains were resistant to colistin and cloxacillin, nearly all were resistant to penicillin, ampicillin and optochine.

The study was carried out on the filtrates of cultures of 60 Bacillus cereus strains isolated from food products which caused food poisoning (20 strains), from patients (20 strains) and from food analysed in routine supervision. The strains isolated from food poisoning demonstrated a more pronounced haemolytic activity as compared with the strains from routine supervision. A lack of correlation was demonstrated between the intensity of lecithinase production and the intensity of reaction of rabbit ileum loop. No strict correlation was noted between the haemolytic properties and lecithinase activity.

In the light of the available literature the present knowledge on the role of Bacillus cereus in alimentary intoxications is discussed, particularly the occurrence and role of B. cereus in food pollution, the most frequent alimentary intoxications in the world are reviewed, the biological tests on animals and studies on humans are described, and the metabolites of the organism are presented. Particular attention is given to the properties of enterotoxins responsible for diarrhoea and vomiting.

Using indirect biological investigations and various experimental models the enterotoxic properties of 60 strains of B. cereus were determined. The determination of biochemical and enzymatic features was insufficient for indicating the enterotoxic strains and differentiating them from the non-enterotoxic ones. In the dermonecrotic test, vascular permeability test and in cultures on cells reliable criteria for determination of enterotoxicity were obtained as confirmed by the response of the ileum. The criterion of enterotoxicity determined in these tests requires raising of the index of ileum pathogenicity to 0.33.