Anatomy and physiology of the autonomic nervous system: Implication on the choice of diagnostic/monitoring tools in 2023.

The autonomic nervous system (ANS) harmoniously regulates all internal organic functions (heart rate, blood pressure, vasomotion, digestive tract motility, endocrinal secretions) and adapts them to the needs. It's the control of so-called vegetative functions, which allows homeostasis but also allostasis of our body. ANS is divided into two systems often understood as antagonistic and complementary: the sympathetic and the parasympathetic systems. However, we currently know of many situations of co-activation of the two systems. Long seen as acting through "reflex" control loops passing through the integration of peripheral information and the efferent response to the peripheral organ, more recent electrophysiological and brain functional imaging knowledge has been able to identify the essential role of the central autonomic network. This element complicates the understanding of the responses of the reflex loops classically used to identify and quantify dysautonomia. Finding the "ANS" tools best suited for the clinician in their daily practice is a challenge that we will attempt to address in this work.

Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.

DNA and heparin alter the internalization process of anti-DNA monoclonal antibodies according to patterns typical of both the charged molecule and the antibody.

The internalization into CHO-K1 fibroblasts of three polyreactive monoclonal IgG2a anti-DNA autoantibodies (mAbs), F14.6, J20.8 and F4.1, isolated from the same unimmunized (NZBxNZW) F1 mouse, and synthetic peptides derived from F4.1 was studied using a technique which quantifies nuclear accumulation. The localization of the mAbs was intranuclear. We compared the influence of two negatively-charged molecules, DNA or heparin. At low concentrations, DNA had dual effects-inhibitory or stimulatory-depending on the mAb. Heparin was inhibitory or had no effect. The possibility that proteoglycans are 'receptors' recognized by anti-DNA mAbs which bind through heparin-sensitive reactions, was explored. Only F4.1 internalization was partly inhibited in glycosaminoglycan-deficient cells. We propose that the complex alterations of internalization patterns of these polyreactive mAbs by the two negatively charged molecules can be explained by (a) the potential of polyreactive mAbs to bind to various charge (or conformation-) dependent 'receptors', (b) the potential of a subclass of mAbs complexed with DNA to utilize additional 'receptor(s)'. Glycosaminoglycans were required for internalization of F4.1-derived peptides, which remained extranuclear, suggesting that nuclear internalization of mAb F4.1 is a multistep process that requires certain sequences present on the intact mAb.

The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.

A mouse homologue of the Saccharomyces cerevisiae Pcd1p coenzyme A diphosphatase, NUDT7alpha, has been expressed as a thioredoxin fusion protein in Escherichia coli. NUDT7alpha is also a CoA diphosphatase of the nudix hydrolase family, and hydrolyses CoA, CoA esters and oxidized CoA with similar efficiences, yielding 3',5'-ADP and the corresponding 4'-phosphopantetheine derivative as products. K(m) and k(cat) values with CoA were 240 microM and 3.8 s(-1). Activity was optimal at pH 8.0 with 5 mM Mg(2+) or Mn(2+) ions, while fluoride was inhibitory with an IC(50) value of 20 microM. Expression of the Nudt7 gene was highest in liver, intermediate in lung and kidney, and lowest in brain and heart, producing a 1.5 kb transcript. A similar pattern of expression was found for the human orthologue, NUDT7. An enzymically inactive splice variant, NUDT7beta, which lacks 20 amino acids downstream of the nudix motif, was also found to be expressed in mouse tissues. Transfection of HeLa cells with a vector expressing the Nudt7alpha gene fused to the C-terminus of red fluorescent protein showed that NUDT7alpha, like Pcd1p, was a peroxisomal enzyme. The function of the NUDT7 enzyme may be the elimination of oxidized CoA from peroxisomes, or the regulation of CoA and acyl-CoA levels in this organelle in response to metabolic demand.

The NADH diphosphatase encoded by the Saccharomyces cerevisiae NPY1 nudix hydrolase gene is located in peroxisomes.

The NPY1 nudix hydrolase gene of Saccharomyces cerevisiae has been cloned and shown to encode a diphosphatase (pyrophosphatase) with NADH as the preferred substrate, giving NMNH and AMP as products. NADPH, diadenosine diphosphate, NAD+, NADP+, and ADP-ribose were also utilized efficiently. Km values for NADH, NAD+, and ADP-ribose were 0.17, 0.5, and 1.3 mM and kcat values 1.5, 0.6, and 0.6 s(-1), respectively. NPY1 has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal (SHL). By fusing NPY1 to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme was found to be targeted to peroxisomes. Colocalization with peroxisomal thiolase was also shown by indirect immunofluorescence. Related sequences in other organisms also have potential PTS1 signals, suggesting an important peroxisomal function for this protein. This function may be the regulation of nicotinamide coenzyme concentrations independently of those in other compartments or the elimination of oxidized nucleotide derivatives from the peroxisomal environment.

The Saccharomyces cerevisiae PCD1 gene encodes a peroxisomal nudix hydrolase active toward coenzyme A and its derivatives.

The PCD1 nudix hydrolase gene of Saccharomyces cerevisiae has been cloned and the Pcd1p protein characterized as a diphosphatase (pyrophosphatase) with specificity for coenzyme A and CoA derivatives. Oxidized CoA disulfide is preferred over CoA as a substrate with K(m) and k(cat) values of 24 micrometer and 5.0 s(-1), respectively, compared with values for CoA of 280 micrometer and 4.6 s(-1) respectively. The products of CoA hydrolysis were 3'-phosphoadenosine 5'-monophosphate and 4'-phosphopantetheine. F(-) ions inhibited the activity with an IC(50) of 22 micrometer. The sequence of Pcd1p contains a potential PTS2 peroxisomal targeting signal. When fused to the N terminus of yeast-enhanced green fluorescent protein, Pcd1p was shown to locate to peroxisomes by confocal microscopy. It was also shown to co-localize with peroxisomal thiolase by immunofluorescence microscopy. N-terminal sequence analysis of the expressed protein revealed the loss of 7 or 8 amino acids, suggesting processing of the proposed PTS2 signal after import. The function of Pcd1p may be to remove potentially toxic oxidized CoA disulfide from peroxisomes in order to maintain the capacity for beta-oxidation of fatty acids.

Cloning, expression and characterization of YSA1H, a human adenosine 5'-diphosphosugar pyrophosphatase possessing a MutT motif.

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5'-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. K(m) and k(cat) values with ADP-ribose were 60 microM and 5.5 s(-1) respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg(2+) or 100-250 microM Mn(2+) ions; fluoride was inhibitory, with an IC(50) of 20 microM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD(+) and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

Efficient gene delivery by a peptide derived from a monoclonal anti-DNA antibody.

We recently reported that translocating murine polyreactive anti-DNA antibodies can be used as vectors for the transfer of macromolecules into cells growing in culture. We show here that two such monoclonal antibodies (J20.8 and F4.1) conjugated to polylysine with a high (93) but not a low (19) number of lysine residues can transfer genes in the presence of serum. A 30 amino acid long peptide, VAYISRGGVSTYYSDTVKGRFTRQKYNKRA (peptide P3), corresponding to joined heavy-chain complementary-determining regions 2 and 3 of F4.1 antibody and carrying 19 lysine residues at its N-terminal, was found to be an efficient vector for the transfection of the luciferase gene into 3T3 and CCL39 cells in the presence of serum. Addition of 0.23 M glycerol during transfection considerably enhanced gene delivery. These results show that conjugation of a short polylysine tail converted a spontaneously internalizing peptide into a potent nontoxic plasmid vector.

The hydrolytic activity of bovine adrenal medullary plasma membranes towards diadenosine polyphosphates is due to alkaline phosphodiesterase-I.

A hydrolase activity directed against diadenosine 5',5"'-P1, P4-tetraphosphate (Ap4A) has been solubilised and partially purified from the plasma membrane fraction of bovine adrenal medullary chromaffin tissue in order to determine its relationship to alkaline phosphodiesterase-I/nucleotide pyrophosphatase (PDase-I, EC 3.1.4.1). Activity with the specific dinucleoside tetraphosphatase (EC 3.6.1. 17) substrate Ap4A and with the non-specific PDase-I substrate thymidine 5'-monophosphate p-nitrophenyl ester had Km and Vmax values of 2.0 microM and 600 pmol/min/mg protein and 0.2 mM and 26 nmol/min/mg protein respectively and co-chromatographed upon gel filtration and ion-exchange chromatography. Activity with the fluorescent substrates etheno-Ap4A and 4-methylumbelliferyl phenylphosphonate co-electrophoresed on native polyacrylamide gels. No activity was detected which exclusively hydrolysed Ap4A. Immunoblotting of the most purified fraction with an antibody against mouse PC-1, one of the major PDase-I family members, detected bands of 240, 120 and 62 kDa corresponding to PC-1 dimer, monomer and proteolytic fragment. Therefore, the activity previously described as bovine adrenal chromaffin cell ecto(diadenosine polyphosphate hydrolase) (ecto-ApnAase) is a PDase-I, probably bovine PC-1.

Stimulation of intracellular Ca2+ levels in human neutrophils by soluble immune complexes. Functional activation of FcgammaRIIIb during priming.

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

Three different genes encode NM23/nucleoside diphosphate kinases in Xenopus laevis.

Nucleoside diphosphate kinases (NDPKs) catalyse the phosphorylation of nucleoside diphosphates. In mammals, the functional enzyme is a hexamer composed of different amounts of two homologous acidic (A) and basic (B) subunits encoded by separate genes. In prokaryotes and invertebrate eukaryotes, only one cytoplasmic enzyme has been isolated. Other genes encoding chloroplastic and mitochondrial forms as well as related proteins have been cloned. Here, we show that in Xenopus laevis, as in mammals, the cytoplasmic NDPK is encoded by several homologous genes. With Xenopus laevis being a pseudotetraploid species, each monomer is encoded by two genes. The amino acid sequences are very similar, and all the differences concern amino acids located at the outer surface of the hexameric enzyme. The Xenopus genes share 82-87% identity with their human counterparts. Interestingly, in vitro, the Xenopus X1 enzyme binds to a specific nuclease hypersensitive element (NHE) of the human c-myc promoter, as does its human counterpart. X1 also binds to a single-stranded (CT)(n) dinucleotide repeat. The NHE is present in the coding strand of a pyrimidine-rich region of the 3' non-coding sequence of the Xenopus NDPK genes. We propose that NDPK is indeed able to bind to its own mRNA and prevent polyadenylation at the normal position. This could provide an autoregulatory translation mechanism. A phylogenetic tree of the vertebrate NDPK sequences supports the idea that in amphibians, as in mammals, gene duplication has resulted in functional diversification.

Diadenosine polyphosphates induce intracellular Ca2+ mobilization in human neutrophils via a pertussis toxin sensitive G-protein.

The diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) and diadenosine 5',5"'-P1,P6-hexaphosphate (Ap6A) all stimulated increases in intracellular Ca2+ in human neutrophils. Maximal increases in intracellular Ca2+ of 650 nM were obtained at dinucleotide concentrations of 500-700 microM. These increases in intracellular, Ca2+ were completely abolished by pre-treatment of the neutrophils with pertussis toxin and were hardly affected when the extracellular buffer was devoid of Ca2+. On the other hand, adenosine triphosphate (ATP) could stimulate much greater increases in intracellular Ca2+ (up to 1.1 microM) at much lower concentrations (half maximal responses obtained at around 5 microM ATP). Receptor de-sensitization experiments indicate that human neutrophils may possess two types of P2-purinoceptors. The first of these may bind ATP (but not the dinucleotides) with high affinity whilst the second may bind the dinucleotides with lower affinity and also bind ATP.

Activation of human neutrophils by soluble immune complexes: role of Fc gamma RII and Fc gamma RIIIb in stimulation of the respiratory burst and elevation of intracellular Ca2+.

Activation of control, unprimed neutrophils with soluble immune complexes fails to generate a respiratory burst. However, if the cells are primed with either tumor necrosis factor-alpha or granulocyte-macrophage colony-stimulating factor prior to addition of soluble immune complexes, then a rapid and transient burst of reactive oxidant secretion is observed. In unprimed neutrophils the soluble immune complexes stimulate an intracellular Ca2+ transient that arises from the mobilization of intracellular Ca2+. However, in primed cells, an "extra" intracellular Ca2+ signal is observed that arises from Ca2+ influx. After removal of Fc gamma RIIIb by treatment with pronase or PI-PLC, the soluble immune complexes fail to activate a respiratory burst in unprimed neutrophils and the "extra" Ca2+ signal is not observed. These results indicate that during priming Fc gamma RIIIb becomes functionally activated and thence its ligation leads to stimulated Ca2+ influx and the generation of intracellular signals that lead to NADPH oxidase activation. Experiments using Fab/F(ab')2 fragments to specifically crosslink either Fc gamma RII or Fc gamma RIIIb and experiments with neutrophils from an individual with Fc gamma RIIIb gene deficiency confirm this important function for Fc gamma RIIIb in neutrophil activation.

Neutrophil apoptosis is delayed by the diadenosine polyphosphates, Ap5A and Ap6A: synergism with granulocyte-macrophage colony-stimulating factor.

In addition to ATP, platelets and other cell types can secrete high quantities of diadenosine polyphosphates Ap3A, Ap4A, Ap5A and Ap6A. There is increasing evidence to show that these molecules can function as novel modulators of cell function. For this report we have measured the effects of the diadenosine polyphosphates Ap5A and Ap6A on neutrophil apoptosis. These molecules can themselves delay neutrophil apoptosis (as assessed by morphology, function. CD16 expression and chromatin integrity), and are as effective on a molar basis as ATP, Ap3A and Ap4A. Moreover, these dinucleotides act synergistically with granulocyte-macrophage colony-stimulating factor (GM-CSF) to delay neutrophil apoptosis. Thus, diadenosine polyphosphates may act, in concert with cytokines, as novel modulators of neutrophil function and survival in certain types of inflammatory conditions.

The diadenosine polyphosphates Ap3A and Ap4A and adenosine triphosphate interact with granulocyte-macrophage colony-stimulating factor to delay neutrophil apoptosis: implications for neutrophil: platelet interactions during inflammation.

Incubation of neutrophils with cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) delays their loss of function and changes in cellular morphology that are characteristic of apoptosis. Adenosine triphosphate (ATP) and the diadenosine polyphosphates Ap4A and AP3A were almost as effective as GM-CSF in delaying neutrophil apoptosis. The nucleotides could thus preserve cellular morphology, protect against chromatin fragmentation, and preserve functions such as NADPH oxidase activity and expression of CD16. Moreover, addition of ATP, AP3A and AP4A together with GM-CSF resulted in more pronounced protection from apoptosis than was observed during incubation with either the cytokine or the nucleotides alone. Because ATP, Ap3A, and AP4A may be secreted from activated platelets, these observations suggest that platelet-derived products, perhaps acting in combination with endothelial-derived or immune cell-derived cytokines, can regulate neutrophil function during certain types of inflammation.

Priming of the respiratory burst of human neutrophils by the diadenosine polyphosphates, AP4A and AP3A: role of intracellular calcium.

The diadenosine polyphosphates, Ap3A and Ap4A, prime the respiratory burst of human neutrophils after stimulation with fMet-Leu-Phe. Maximal priming of oxidase activity occurred at 600-800 microM Ap3A and Ap4A, compared with maximal priming observed at 200 microM ATP. The time course of priming of the oxidase by all 3 nucleotides was very rapid, being detectable if added within 10 s of fMet-Leu-Phe. All 3 nucleotides also elicited increases in intracellular Ca2+ levels and there was a close concentration-dependency between the extent of priming and the increase in intracellular Ca2+. However, at low concentrations of nucleotides (< 50 microM Ap3A and Ap4A and < 0.1 microM ATP) priming of the oxidase was observed without detectable increases in intracellular Ca2+. These observations indicate that diadenosine polyphosphates may be novel regulators of neutrophil function and that priming of oxidase activity may occur via mechanisms that are either dependent or independent of increases in intracellular Ca2+.

The effects of staurosporine and okadaic acid on baby hamster kidney fibroblast cell adhesion.

The effects of staurosporine, a potent protein kinase C inhibitor, and okadaic acid, a non-TPA tumour promoter, on the adhesion of BHK fibroblast were investigated. Staurosporine at 2.5 and 5 microM was found to stimulate a gradual increase in BHK cell adhesion as well as spreading in 3% serum-containing medium. An increase of approximately 27% over the control value was found at 5 microM concentration in 20 minutes. No such effect was seen in serum-free conditions. Staurosporine at 5 microM, enhanced BHK cell-cell adhesion in 3% serum and in serum-free conditions. Okadaic acid, a phosphatase inhibitor, at concentrations between 0.25 and 1 microgram/ml, was found to inhibit BHK cell-substratum adhesion and spreading. The inhibitory effect was time and concentration dependent. These findings suggest that protein kinase C might be involved in the mechanism(s) controlling BHK cell attachment.

Localised application of an activating signal to a cell: experimental use of fibronectin bound to beads and the implications for mechanisms of adhesion.

Small beads derivatised with fibronectin or with bovine serum albumin are allowed to attach to BHK cells in suspension at low ratios of beads to cells. In this way populations of cells bearing predominantly one bead per cell can be prepared. We show that the attachment of one bead per cell affects the adhesion and spreading of that cell on substrata, raising adhesion and increasing spreading if the signal molecule is fibronectin, decreasing these quantities if the bead bears BSA. The experiments are conducted in the absence of other sources of exogenous fibronectin and in some cases in the additional absence of endogenous sources. The effects are especially marked if the substratum is adsorbed haemoglobin on which control cells show little attachment or spreading. We further show by interference reflection microscopy and by scanning electron microscopy that the beads are found on the non-adhering side (uppermost or outer) of the cell when fibronectin-bearing beads are used, presumably because fibronectin will not attach to haemoglobin. The increased adhesion and spreading found in such cases must be attributed to an activation produced by the bead, which spreads to other parts of the cell and which activates a fibronectin-independent mode of adhesion.