The Role of Baroreflex Sensitivity in Acute Hypotensive Episodes Prediction in the Intensive Care Unit.

A life threatening condition in Intensive Care Unit (ICU) is the Acute Hypotensive Episode (AHE). Patients experiencing an AHE may suffer from irreversible organ damage associated with increased mortality. Predicting the onset of AHE could be of pivotal importance to establish appropriate and timely interventions. We propose a method that, using waveforms widely acquired in ICU, like Arterial Blood Pressure (ABP) and Electrocardiogram (ECG), will extract features relative to the cardiac system to predict whether or not a patient will experience a hypotensive episode. Specifically, we want to assess if there are hidden patterns in the dynamics of baroreflex able to improve the prediction of AHEs. We will investigate the predictive power of features related to the baroreflex by performing classifications with and without them. Results are obtained using 17 classifiers belonging to different model families: classification trees, Support Vector Machines (SVMs), K-Nearest Neighbors (KNNs) replicated with different set of hyper-parameters and logistic regression. On average, the use of baroreflex features in the AHE prediction process increases the Area Under the Curve (AUC) by 10%.

The proton pump interactor (Ppi) gene family of Arabidopsis thaliana: expression pattern of Ppi1 and characterisation of knockout mutants for Ppi1 and 2.

Plant plasma membrane H+-ATPases (PM H+-ATPase) are essential for establishing a proton electrochemical gradient across the cell plasma membrane. Their regulation is poorly understood, except for the role of 14-3-3 proteins, which relieve autoinhibition from the C-terminal domain. A novel protein interacting with this domain was recently identified in Arabidopsis and named PPI1 (Proton Pump Interactor 1). PPI1 stimulates PM H+-ATPase activity in vitro. Here, we analyse the expression pattern of Ppi1 using beta-glucuronidase as a reporter. Expression is strong in root and shoot vascular systems, particularly in meristematic and sink tissues, as well as in pollen, stigmas and siliques, but not in developing embryos. Removal of the first intron decreased GUS expression 45-fold. We also analysed the transcription of Ppi2, another gene in the family, and demonstrated that Ppi2 is expressed in seedlings, cultured cells and flowers. We reassessed Ppi2 gene structure based on RT-PCR amplifications, cDNA data and similarity to other Ppi genes. Insertional mutants for both Ppi1 and Ppi2 were isolated. Two different mutants of Ppi1 showed aberrant mRNAs and lacked any detectable protein and are therefore true knockouts. Interestingly, one mutation inhibited the splicing of one intron at a considerable distance (>700 bp) from the T-DNA insertion site, but not the splicing of a proximal intron (29 bp) or of any other intron. At the plant level, neither of the single mutants nor the double ppi1ppi2 mutant showed an altered phenotype in standard growth conditions under acid load or salt stress.

At-ACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus.

A Ca(2+)-ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca(2+)-ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca(2+)-transporting P-type Ca(2+)-ATPases. The identity of At-ACA8p as the PM Ca(2+)-ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca(2+)-ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM localized Ca(2+)-ATPase to be cloned and is clearly distinct from animal PM Ca(2+)-ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence of At-ACA8p and those of endomembrane-localized type IIB Ca(2+)-ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca(2+)-ATPases.

A non-destructive selection method for resistance to fusicoccin in Arabidopsis thaliana.

A mutagenised population of seeds of Arabidopsis thaliana was allowed to germinate in the presence of the positively charged aminoglycoside hygromycin (4 µg/ml) and the fungal toxin fusicoccin (5×10-6 M). This hygromycin concentration, which is non-toxic by itself, becomes toxic when used together with fusicoccin, which stimulates cation uptake. Seeds that had germinated after 3-5 days and produced seedlings with green cotyledons were potentially resistant to fusicoccin and were therefore transferred into sterile Magenta vessels containing 1/2-strength Murashige and Skoog medium. This selection procedure is non-destructive, i.e. it allows the recovery of viable seedlings and their growth into adult plants thus permitting direct physiological characterisation.

Complementation of a yeast delta pkc1 mutant by the Arabidopsis proteinANT.

The Saccharomyces cerevisiae protein kinase C homologue, PKC1, is involved in maintenance of cell integrity during polarized growth. We have used a mutant complementation approach to investigate related signal transduction pathways in higher plants. Here we report the isolation of a cDNA from Arabidopsis thaliana which partially suppresses the lytic defect of a delta pkc1 yeast strain. The encoded protein, ANT, belongs to the AP2-related gene family and is essential for ovule development. Expression in yeast of a LexA-ANT fusion protein activates transcription of a reporter gene from promoters containing lexA operators. Our results support the idea that ANT acts as transcriptional activator in planta.

Wild-type strains of Dictyostelium discoideum can be transformed using a novel selection cassette driven by the promoter of the ribosomal V18 gene.

Almost all methods for transformation of the social ameba Dictyostelium discoideum rely on axenic growth, that is, growth in a synthetic medium, for at least part of the procedure. Axenic growth requires several mutations. Here we describe a procedure that can be used to transform wild-type strains which are able to grow only on the natural food source, bacteria. The method relies on a new selection cassette driven by the V18 promoter, a promoter that we show is substantially more active during growth on bacteria than the actin-6 promoter, which is widely used for axenic transformation. The procedure gives transformation frequencies of about 10(-5) with both strains Ax2 (capable of axenic growth) and NC4 (capable of growth only on bacteria). Using this vector, we have obtained NC4 strains carrying several beta-galactosidase reporter cassettes. Our vector can also be used in axenic transformations.

Cloning vectors for the production of proteins in Dictyostelium discoideum.

We constructed and tested a series of cloning vectors designed to facilitate protein production and purification in Dictyostelium discoideum (Dd). These vectors carry the origin of replication of the Dd high-copy-number plasmid Ddp2, expression cassettes consisting of the strong, constitutive actin (act15) or the inducible discoidin (disI gamma) promoters, a translational start codon upstream from a multiple cloning site and sequences for the addition of epitope or affinity tags at the N- or C-termini of any protein. The affinity tag used corresponds to 7 (N-terminal fusion) or 8 (C-terminal fusion) His residues. The epitope tags correspond to an 11-amino-acid sequence from human c-myc, recognised by monoclonal antibody (mAb) 9E10, and the Glu-Glu-Phe sequence recognised by mAb YL1/2 to alpha-tubulin. Both these mAb are commercially available. The YL1/2 epitope offers a second affinity tag for the purification of proteins under native conditions. The functional competence of the vectors was tested by determining their ability to promote the expression of various Dd myosin constructs. High synthesis levels were obtained for each vector; up to 1 mg of homogenous, functional protein per g of cells was obtained after purification of the recombinant products.

An actin-related protein from Dictyostelium discoideum is developmentally regulated and associated with mitochondria.

An actin-related protein (ACLA) has been identified in the cellular slime mould Dictyostelium discoideum. The complete cDNA sequence indicates that within the actin superfamily it belongs to the ARP3 family of actin-related proteins together with Arp66B from Drosophila melanogaster, Actin2 from Bos taurus, act2 from Schizosaccharomyces pombe and possibly act2 from Caenorhabditis elegans. The ACLA mRNA is regulated during development, showing a maximum between 2 and 4 h after starvation. The protein has been expressed in E. coli and antibodies raised against it. Immunofluorescence microscopy shows that ACLA protein co-localises with mitochondria; the protein copurifies with Dictyostelium mitochondria.

The proximal pathway of metabolism of the chlorinated signal molecule differentiation-inducing factor-1 (DIF-1) in the cellular slime mould Dictyostelium.

Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s., n.m.r. and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties of a cytochrome P-450. It is membrane-bound and uses NADPH as co-substrate. It is also inhibited by CO, the classic cytochrome P-450 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, with the exception of the DIF-3 analogue lacking the chlorine atom. The Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development.

The 'prespore-like cells' of Dictyostelium have ceased to express a prespore gene: analysis using short-lived beta-galactosidases as reporters.

In transgenic strains of Dictyostelium discoideum that express beta-galactosidase under the control of a prespore-specific promoter, only early slugs show reporter confined to the prespore zone. As slugs migrate beta-galactosidase-positive cells accumulate in the prestalk zone; ultimately, there may be so many that the prestalk-prespore boundary is no longer distinguishable (Harwood, A., Early, A., Jermyn, K. and Williams, J. (1991) Differentiation 46, 7-13). It is not clear whether these 'anomalous' reporter-positive cells currently express prespore genes; another possibility is that they are ex-prespore cells that have transformed to prestalk and sorted to the prestalk zone (Sternfeld, J. (1993) Roux Archiv. Dev. Biol. 201, 354-363), while retaining their previously produced reporter. To test the activity of the prespore genes in these cells, we have made prespore reporter constructs whose products decay quickly; these are based on constructs used to investigate protein turnover in yeast (Bachmair, A., Finley, D. and Varshavsky, A. (1986) Science 234, 179-186). In strains bearing such constructs, beta-galactosidase-positive cells do not appear in the prestalk zone. The apparent deterioration of the prestalk/prespore pattern in older slugs is thus an artefact of reporter stability.

Mutants of Dictyostelium discoideum with defects in the regulation of discoidin I expression.

Discoidins are proteins, coded by a multigene family, which are regulated by extracellular factors during growth and development of Dictyostelium discoideum. In this paper we describe the isolation and characterization of mutants which misregulate the expression of the discoidin I subgroup. One mutant (III29) induces discoidin I during late growth phase but does not express it during development. Another mutant (VI41) has significantly reduced discoidin levels under all conditions tested, while two mutants (VI88 and X27) express discoidin early during exponential growth and accumulate more discoidin protein than the wild type. The defects are due to abnormal regulation of transcription in all mutants except VI41. Experiments in which mutants and wild type are mixed suggest that the mutant phenotypes are not caused by changes in extracellular signals. Since multiple members of the multigene family are affected, it can be concluded that the intracellular signals regulating discoidin expression are changed rather than the genes themselves. The mutants are thus likely to have defects in the reception or intracellular processing of environmental signals.

Transcriptional regulation by folate: inducible gene expression in Dictyostelium transformants during growth and early development.

The Dictyostelium discoidin genes are induced in bacteria-grown cells shortly before the onset of development but are also highly expressed during growth in axenic medium. We here show that axenically growing cells strongly respond to the extracellular signal folate by suppressing discoidin synthesis while cell growth and development is not substantially affected. Repression occurs via two previously identified promoter elements, the dIE and the dAXE. Removal of the signal molecules or setting cells up for development results in rapid reactivation of the promoter. Based on this observation, we constructed the transformation vector pVEII and describe a convenient method which allows for controlled expression of a gene of interest in growing cells and also for external modulation in early development. Deletion constructs of the discoidin promoter can be used in addition to vary transcriptional activity over about one order of magnitude.

Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP.

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.

Analysis of specific mRNA destabilization during Dictyostelium development.

A number of specific mRNAs are destabilized upon disaggregation of developing Dictyostelium discoideum cells. Analysis of a family of cloned genes indicates that only prespore-enriched mRNAs are affected; constitutive mRNAs that are expressed throughout development and mRNAs that accumulate preferentially in prestalk cells are stable under these conditions. The decay of sensitive prespore mRNAs can be halted by allowing the cells to reaggregate, indicating that destabilization occurs by the progressive selection of individual molecules rather than on all members of an mRNA subpopulation at the time of disaggregation. Individual molecules of the sensitive mRNA species remain engaged in protein synthesis in the disaggregated cells until selected. Destabilization of sensitive mRNAs is induced by cell dissociation even in the presence of concentrations of nogalamycin that inhibit RNA synthesis. The reported prevention of disaggregation-induced mRNA decay by actinomycin D and daunomycin is therefore probably a secondary effect unrelated to the inhibition of transcription.