Detection of stress and the effects of central nervous system depressants by gastrointestinal smooth muscle electromyography in wakeful rats.

AIMS: The altered gut-brain interaction can be in the background of functional gastrointestinal (GI) disorders. In the GI tract, the slow-wave myoelectric signals can be detected by electromyography (EMG). The aims of our study were to follow up the stress induced alteration in the GI tract by smooth muscle EMG in wakeful rats. MAIN METHODS: The GI tract myoelectric activity of male rats was measured by an electrode pair under the abdominal skin, the responses were detected and analyzed by a software using fast Fourier transformation. Animals were immobilized and treated with either diazepam or haloperidol. The plasma corticosterone level was determined by ELISA kit, the levels of drugs were measured by HPLC, while the direct GI effects of the compounds were tested in an organ bath. KEY FINDINGS: Significant correlation (r2 = 0.52) was found between the immobilization induced increase in the EMG spectra of the GI tract segments and the increase in corticosterone plasma levels. The stress-reducing effects of diazepam and haloperidol were also detectable by smooth muscle EMG in the GI tract. No direct smooth muscle actions of the drugs were found in organ bath studies. SIGNIFICANCE: The smooth muscle EMG instrument can measure the level of acute stress and is applicable for the investigation of central nervous system affecting drugs through the GI tract in awake rats. This is the first tool to measure the stress response via the GI tract reactions. The technique may open a new perspective in the diagnosis and therapy of psychosomatic disorders.

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse.

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.

Abnormal cell-specific expressions of certain protein kinase C isoenzymes in peripheral mononuclear cells of patients with systemic lupus erythematosus: effect of corticosteroid application.

We have studied the expressions of various protein kinase C (PKC) isoenzymes in T cells and monocytes from patients with systemic lupus erythematosus (SLE), in comparison to those of healthy controls and patients with other immunological disorders. As measured by Western blotting, the levels of PKCbeta, delta, eta, epsilon, theta and zeta (but not of PKCalpha) significantly decreased in T cells of SLE patients. In monocytes, however, we observed marked suppressions only in the expressions of PKCdelta, epsilon and zeta but not in the expressions of other PKC isoforms. In vivo corticosteroid application, as well as in vitro steroid treatment of monocytes, elevated the expressions of most isoforms close to normal values; however, the decreased levels of PKCtheta and zeta were not affected by steroid application. These alterations were characteristic to SLE because we could not detect any changes in the PKC levels in mononuclear cells of primary Sjögren's syndrome and mixed connective tissue disease patients. These results suggest that impaired PKC isoenzyme pattern may exist in the T cells and monocytes of SLE patients. Furthermore, the clinically efficient glucocorticoid application in SLE can increase the expression of some members of PKC system.

A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells.

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.

Decreased arachidonic acid release in peripheral blood monocytes of patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the release of arachidonic acid (AA) in unfractionated peripheral blood mononuclear cells (PBMC), separated monocytes and T lymphocytes of patients with systemic lupus erythematosus (SLE). METHODS: AA release was measured in cells from 56 patients with SLE and from 48 controls. Of the 56 patients with SLE, 38 were receiving glucocorticosteroids and 18 were not. [3H]AA was incorporated into the membranes of PBMC and purified subsets of monocytes and T lymphocytes. The release of [3H]AA was measured both in nonstimulated cells cultured for 24 h and in cell cultures stimulated by phorbol ester (PMA) and Ca2+ ionophore for 4 h. RESULTS: In the PBMC of SLE patients not taking glucocorticosteroids, the release of AA was decreased in both stimulated and nonstimulated cells. There was a decrease of AA production in monocytes but not in T lymphocytes. This phenomenon could be observed in the active and inactive phases of the disease. CONCLUSION: A defect in AA production may exist in the peripheral monocytes of patients with SLE, resulting in decreased release of AA in patients not receiving glucocorticosteroid therapy.

Urinary findings in asymptomatic subjects with spina bifida treated with intermittent catheterization.

The urines from 43 asymptomatic children with spina bifida were examined. Eighty-one percent were abnormal because of bacteriuria and pyuria (51%), bacteriuria alone (26%) or pyuria alone (5%). Interleukin-8 was elevated in 54% of the abnormal urines. The presence of pyuria and interleukin 8 suggests that the asymptomatic bacteriuria reflects low grade infection rather than colonization.

Glucocorticosteroid dependent decrease in the activity of calcineurin in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus.

OBJECTIVE: To compare the activity of calcineurin in the peripheral blood mononuclear cells (PBMC) of 32 patients with systemic lupus erythematosus (SLE) and 35 healthy controls. METHODS: The activity of calcineurin was assayed in the supernatants of sonicated mononuclear cells. RESULTS: There was no significant difference in the calcineurin activity of patients with SLE not taking glucocorticosteroids (GCS) compared with the healthy controls. On the other hand, the activity of calcineurin was reduced in patients with SLE taking GCS, correlating negatively with the dose of GCS. The stimulation of PBMC by phorbol ester and calcium ionophore decreased the calcineurin activity both in patients with SLE and in healthy controls. GCS could also reduce calcineurin activity in the mononuclear cells of healthy subjects in vitro. CONCLUSIONS: In patients with SLE the decrease in the calcineurin activity of PBMC depended on the dose of GCS used for treatment, and it was not a disease specific alteration. The higher the dose of GCS, the greater the inhibition of calcineurin activity. The reduction of calcineurin activity is a new element in the immunosuppressive effects of GCS during the treatment of patients with SLE.

Comparative study on the effect of phosphorylated TCR zeta chain ITAM sequences on early activation events in Jurkat T cells.

One of the main dilemma in T cell receptor (TCR) signal transduction is whether the presence of multiple Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) within the TCR signaling module serves for signal amplification or signal distribution. To contribute to answer this question, we analyzed the effect of synthetic oligopeptides representing the three bi-phosphorylated zeta chain-ITAMs on the early signaling events in permeabilized leukemia T cells. Our main observations were as follows: 1/Stimulation of the cells with the bi-phosphorylated membrane proximal and central ITAMs (zeta (1)y(p)y(p) and zeta (2)y(p)y(p), respectively) resulted in a strong phosphorylation of proteins with a similar pattern. In contrast, the membrane distal ITAM, zeta (3)y(p)y(p) had a reduced ability to promote tyrosine phosphorylation and failed to induce the phosphorylation of a number of proteins. 2/ The phospho-peptide induced tyrosine phosphorylation events were at least partially mediated by p56(lck) and Syk/ZAP70 protein tyrosine kinases as it was shown in p56(lck) and Syk/ZAP70 deficient Jurkat variants. 3/The patterns of the association of the adaptor protein, Grb2 with tyrosine phosphorylated proteins following cell stimulation with the bi-phosphorylated membrane proximal or the central ITAMs were similar, while the membrane distal ITAM was unable to induce any of these associations. Our data provide additional evidence that the three zetaITAMs differ in their capacity to induce tyrosine phosphorylation of intracellular proteins in permeabilized T cells, depending to their primary sequence. The first and second ITAM sequences of the zeta chain may have similar but not totally overlapping functions. This conclusion results from their similar but not identical abilities to induce tyrosine phosphorylation and association of Grb-2 with intracellular phosphoproteins. In contrast, the third ITAM (zeta3) may have distinct functions since this peptide fails to induce tyrosine phosphorylation of a number of proteins compared to the other two ITAMs, and it is unable to induce either new association or the increase in the amount of Grb-2 associated phosphoproteins.

Inhibition of calcineurin activity and protection against cyclosporine A induced cytotoxicity by prednisolone sodium succinate in human peripheral mononuclear cells.

We have investigated the effects of prednisolone sodium succinate (Pss) and cyclosporin A (CSA), applied alone or concurrently, on the release of arachidonic acid (AA) (cytosolic phospholipase A(2) (cPLA(2)) activity) and on the calcineurin (CN) activity of human peripheral blood mononuclear cells (PBMC). The cytotoxic damage to the cells treated by the drugs was estimated by the release of lactate dehydrogenase (LDH). We found that Pss (10(-5) M) could inhibit the CN activity and higher concentrations (10(-4) M) could decrease the cytotoxic damage caused by CSA (10(-4) M) during their combined application. CSA had no specific effect on the release of AA from the cells. In the combined clinical use of glucocorticosteroids (GCS) and CSA, their additive inhibitory effect on CN activity and the protective membrane influence of GCS against the cytotoxicity of CSA may be beneficial.

Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2.

The C-terminal domain of microtubule-associated protein 2 (MAP2) contains a proline-rich region and the tubulin-binding domain. We have generated antibodies to follow the phosphorylation state of the proline-rich domain. One of these antibodies (no. 305) has been raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the threonine residues. This sequence is present in the proline-rich region of MAP2 and is phosphorylated in vitro by at least three different proline-directed protein kinases: p42mpk, p34cdc2, and GSK3 (glycogen-synthase kinase 3) alpha/beta. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as determined by Staphylococcus aureus V8 protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all three kinases studied with similar efficiency. In high-molecular-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.

Dephosphorylation of tau protein from Alzheimer's disease patients.

Tau protein-prepared post-mortem from brains of Alzheimer's disease patients was treated with protein phosphatase 1 catalytic subunit, 2A catalytic subunit, and 2B (calcineurin). Dephosphorylation was monitored by immunoblotting with two monoclonal antibodies, TAU-1 and SMI31, which recognize in tau the dephospho- and phospho-states, respectively, of proline-directed protein kinase phosphorylation sites. Out of the three enzymes tested, protein phosphatase 2A was only effective in dephosphorylating tau at these Alzheimer-type epitopes.

Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate.

We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.

Dephosphorylation of distinct sites on microtubule-associated protein MAP1B by protein phosphatases 1, 2A and 2B.

Rat brain microtubule-associated protein MAP1B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAP1B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline-directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.

Synthesis and enzymatic activity of some new purine ring system analogues of adenosine 3',5'-cyclic monophosphate.

A series of novel adenosine 3',5'-cyclic monophosphate (cAMP) analogues, as well as their 6-deamino and 6-nitro derivatives, were synthesized where the purine ring was replaced by indazole, benzotriazole, and benzimidazole. The 3',5'-cyclic monophosphates of indazole and benzotriazole ribofuranosides, where the sugar-phosphate moiety is attached to the N-2 nitrogen atoms of the heterocycles, were also prepared. The biological efficiency of the analogues was tested by their ability to activate purified cAMP-dependent protein kinase I (PK-I) from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart. Each cyclic nucleotide is capable of activating both PK isozymes in half-maximum concentrations (Ka) ranging from 2.0 x 10(-8) to 4.8 x 10(-6) M. The cyclic phosphate of N-1-beta-D-ribofuranosylindazole (13) proved to be a very poor activator for both PK-I and PK-II, but when indazole binds by N-2 to ribose or when the hydrogen atom at C-4 is substituted by a nitro or amino group, activities of the analogues increase considerably. The activating potencies of benzotriazole derivatives are similar to that of cAMP, irrespective of the C-4 substituents. The Ka' values of cyclic nucleotides containing benzimidazole were found to be higher for PK-II than for PK-I; e.g. the activity of 4-nitro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphate (32) is nearly 20 times as high for PK-II than for PK-I.

Comparative characterization of liver glycogen metabolism in rat and guinea-pig.

1. Guinea-pig liver contained more phosphorylase in the active (phosphorylated) form and less synthase in the active (dephosphorylated) form when compared with rat liver. 2. Activities of cyclic AMP-dependent protein kinase and Ca(2+)-dependent phosphorylase kinase were the same in rat and guinea-pig livers. 3. Activities of phosphorylase phosphatase and synthase phosphatase in the extract and glycogen plus microsomal fraction of guinea-pig liver were significantly lower than those of rat liver. 4. The existence of inhibitor-1 in the liver of guinea-pig can maintain a lower activity of type-1 protein phosphatase, especially when inhibitor-1 is phosphorylated by cyclic AMP-dependent protein kinase.

Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.

Formation of partially phosphorylated phosphorylase in isoproterenol stimulated rat hearts.

Phosphorylase ab hybrid was demonstrated in perfused rat hearts and during the in vitro conversion of purified rat heart phosphorylase b. Phosphorylase ab hybrid was determined in rat heart extracts by the activating effect of AMP in the presence of caffeine. These results were confirmed by the quantitative determination of incorporated 32P in vitro and through the characteristic inhibition of ab hybrid by glucose-6-phosphate. As shown by our results, in aerobically perfused control hearts only the ab hybrid represents the active form of phosphorylase, its activity reaching about 20% of the total. In response to isoproterenol (5-1000 ng), the amount of ab hybrid rose to about 30-40%, preceding the rise of the a form, which increased in a dose-dependent manner up to 45% of the total. The great sensitivity of the ab form to AMP activation and glucose-6-phosphate inhibition supports its physiological significance in heart under in vivo conditions as well. Our results strongly suggest that the activity ratio -AMP/ + AMP reflects rather the percentage ratio of phosphorylated subunits than that of the activated (partially or totally phosphorylated) phosphorylase molecules.