Erythrocyte glutathione transferase: a non-antibody biomarker for systemic sclerosis, which correlates with severity and activity of the disease.

Erythrocyte glutathione transferase (e-GST) is a detoxifying enzyme hyper-expressed in nephropathic patients and used recently as a biomarker for blood toxicity. Systemic sclerosis (SSc) is characterized by endothelial dysfunction and fibrosis of the skin and internal organs. Renal involvement is frequent in SSc patients. Here we show that e-GST is hyper-expressed in SSc patients (n = 102) and correlates (R(2) = 0.49, P < 0.0001) with the Medsger DSS and DAI Valentini indices that quantify the severity and activity of this disease. Interestingly, e-GST does not correlate with the impairment of kidney or other specific organs taken separately. e-GST hyper-expression seems to be linked to the presence of a factor (i.e., toxin) that triggers the autoimmune disease, and not to the damage of specific organs or to oxidative stress. e-GST may be proposed as an innovative non-antibody biomarker for SSc useful to check the progress of this disease and the efficiency of new therapeutic strategies.

In vitro and in vivo phase II metabolism of combretastatin A-4: evidence for the formation of a sulphate conjugate metabolite.

1. Combretastatin A-4 (CA-4), is a natural compound with a potent tubulin polymerization and cell growth inhibitor properties. For these reasons CA-4 is one of the most potent anti-vascular agents that shows strong cytotoxicity against a variety of human cancer cells, including multi-drug-resistant cancer cell lines. In order to complete the knowledge of metabolic fate of CA-4, the in vitro and in vivo phase II metabolism was investigated. 2. Both in incubation with rat and human liver S9 preparation in the presence of 39-phosphoadenosine-5 -phosphosulfate (PAPS) as a cofactor the formation of a previously no reported sulphate metabolite was demonstrated through liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) data and comparison with a synthetic reference sample. 3. In incubation of CA-4 using rat and human liver microsomes, the formation of CA-4 glucuronide was observed and chromatographic and mass spectral properties of the metabolite were achieved and compared with those of a synthetic reference sample. 4. Incubation of CA-4 with rat and human liver S9 preparation in the presence of uridine-5 -diphosphoglucuronic acid trisodium salt (UDPGA) and an beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system as cofactors resulted in the formation of glucuronides arising from phase I CA-4 metabolites. 5. When CA-4 was administered intraperitoneally to rat at a dose of 30 mg kg(-1), both glucuronide and sulphate metabolites were observed in LC-ESI-MS-MS chromatograms and their mass spectral data were identical to those obtained from synthetic standards.

Balance between autocrine interleukin-1beta and caspases defines life versus death of polymorphonuclear cells.

The role of endogenous IL-1beta in regulating spontaneous and Fas-triggered apoptosis of human PMN has been studied in relation to the activity of the IL-1beta-generating enzyme ICE (caspase-1), an enzyme also involved in the mechanism of cell death. Upon in vitro culture, PMN undergo spontaneous apoptosis and express increasing levels of IL-1beta, caspase-1- and caspase-3-like enzymes. Endogenous IL-1beta protects PMN from apoptosis, since inhibition of either IL-1beta or caspase-1 activity can accelerate PMN apoptotic death. Thus, in spontaneous PMN apoptosis caspase-1 essentially plays an anti-apoptotic role by inducing maturation of protective IL-1beta, whereas other molecules are responsible of driving apoptosis. Upon Fas triggering, PMN apoptosis is greatly accelerated, in correlation with increased caspase activity, whereas IL-1beta production is not augmented. Inhibition of IL-1beta activity can increase Fas-induced apoptosis, whereas caspase-1 inhibitors are without significant effect. It is hypothesized that in Fas-induced PMN apoptosis caspase-1 has a double role: it can protect from apoptosis through generation of protective IL-1beta, as in spontaneous apoptosis, and it can also exert pro-apoptotic activity which counterbalances the protective effect and allows accelerated apoptosis.

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II.

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.

Involvement of an arachidonic-acid-dependent pathway in the interferon-beta-mediated expression of C202 gene in Ehrlich-ascites-tumor cells.

We have investigated the signal transduction mechanism of the expression of the C202 gene mediated by interferon beta (IFN-beta) in the murine Ehrlich's ascites tumor cell line. We have shown that treatment of cells with IFN-beta transiently enhances within minutes the release of free arachidonic acid through membrane phospholipase activity. Furthermore, prior treatment with either p-bromophenacyl bromide, an antagonist of both cytosolic and secretory phospholipase A2, or neomycin, which blocks phospholipase C activity, significantly decreased the activation of the murine IFN-beta-inducible gene, C202. Moreover, an increase of the expression of the C202 gene was observed after blocking of both the cyclooxygenase and lipoxygenase pathways. This suggests that further metabolism of arachidonic acid to epoxides via epoxygenase-catalysed pathways may be a mechanism by which second messengers for IFN-beta-mediated effects on C202 gene expression are generated. Taken together, these results indicate that lipids as second messengers may be important mediators in the IFN-beta-based activation of C202 gene expression.

Mapping of receptor binding sites on IL-1 beta by reconstruction of IL-1ra-like domains.

Upon structure comparison between IL-1 beta and its antagonist IL-1ra, single or multiple residues along the IL-1 beta sequence were replaced with the corresponding amino acids present in the IL-1ra protein, in the attempt to identify sites important for receptor binding and for biologic activity on the two molecules. Ten of fifteen mutant proteins had activity comparable to that of wild-type IL-1 beta in three different biologic assays and in receptor binding, indicating that the introduced changes did not influence the functional structure of the protein. Conversely, three mutants (SMIL-9: 127/263 R/T-->W/Y; SMIL-10: 125/127/263/265 T/R/T/Q-->R/W/Y/E; SMIL-15:222/227 I/E-->S/S) showed an increased binding capacity for IL-1RI, not paralleled by increased agonist activity, indicating that the introduced IL-1ra residues could be involved in the nonagonist IL-1RI binding site. On the other hand, two mutants showed diminished binding capacity with concomitant decrease in biologic activity. Both mutants (SMIL-1, five substitutions in the loop 202-214; and SMIL-3, total replacement of the loop 164-173 with the IL-1ra stretch 52-55) included substitutions of residues allegedly important for agonist binding to IL-1RI. Mutant SMIL-3 showed the most profound reduction in binding capacity for IL-1RI (CDw121a) and a more than 1,000-fold reduced biologic activity both in vitro and in vivo, but it retained full capacity of binding to IL-1RII (CDw121b) and acted as a selective antagonist of IL-1RII. From these results the following conclusions can be drawn. IL-1 beta binds to IL-1RI and to IL-1RII through different sites, and the loop 164-173 appears as one of the areas involved in the selective interaction with IL-1RI. Agonist (IL-1 beta) and nonagonist (IL-1ra) binding to IL-1RI occur through distinct sites, with loops 164-173 and 202-214 of IL-1 beta identified as two of the sites selectively involved in agonist binding to the activating receptor.

Laparoscopic cholecystectomy: imaging of complications and normal postoperative CT appearance.

Six patients underwent imaging studies to evaluate complications related to laparoscopic cholecystectomy. In addition, computed tomography (CT) of the abdomen and pelvis was performed on six patients 3-5 days after uncomplicated laparoscopic cholecystectomy in order to further clarify the normal postoperative CT appearance in these patients. Complications included ureteral laceration with periureteric hematoma and ureteroperitoneal fistula, hepatic artery pseudoaneurysm, hepatic laceration, retained common bile duct stone, bile leak, and biloma of the abdominal wall. At 3-5 days following uncomplicated laparoscopic cholecystectomy, typical CT findings include fluid density in the gallbladder fossa, a very small amount of pelvic fluid, and small densities within the subcutaneous fat at the expected sites of trocar insertion.

Involvement of phospholipase A2 activation and arachidonic acid metabolism in the cytotoxic functions of rat NK cells.

Arachidonic acid (AA) release via phospholipase A2 (PLA2) activation and generation of eicosanoids have been implicated as playing signaling roles in a variety of cell types. Here we show evidence that interaction of fresh NK cells with membranes from sensitive or antibody (Ab)-coated targets generates eicosanoids through both cyclooxygenase (CO) and lipoxygenase (LO) pathways. Eicosanoid generation is attributable to PLA2 activation since pretreatment with PLA2 irreversible inhibitors, such as mepacrine or para-bromophenacylbromide (pBPB), completely blocks AA metabolite generation. The involvement of PLA2 or AA metabolites in the cytotoxic functions of rat NK cells has also been investigated. Treatment of effector cells with mepacrine or pBPB resulted in complete, irreversible, dose-dependent inhibition of both NK and ADCC activities, which were completely reversed by the addition of exogenous PLA2 or its hydrolysis products, AA and lysophosphatidylcholine (lysoPC). Among the metabolites of AA released by NK cells, the 5-LO product leukotriene B4 (LTB4) seems to play an important role in cytolytic activities of NK cells. Indeed, the LO inhibitor, nordihydroguaiaretic acid (NDGA), totally abrogated both NK and ADCC activities, which were restored by the addition of exogenous LTB4. However, the failure of LTB4 to reverse mepacrine or pBPB-induced inhibition of NK and ADCC suggests that its effects could be mediated by PLA2. The results are consistent with a crucial role for the target-stimulated AA release as a fundamental step in the signal transduction pathway in NK cell. Moreover, LTB4 generation seems to be responsible for further PLA2 activation in a second step leading to the amplification of response.

Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation.

CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.

A possible plasma membrane particle containing malic enzyme activity.

A definite membrane fraction from Cucurbita hypocotyls, maize coleoptiles, and other plant tissues contains a NADP-dependent malic enzyme activity, up to 10% of overall tissue activity, and probably other soluble proteins. This "malic enzyme particle" is identified as plasmalemma on the basis of sedimentation behavior, density distribution in sucrose gradients, in comparison with enzyme markers, and sluggish penetration by the sugar Metrizamide. Enzyme binding to the plasma membrane is stable and scarcely sensitive to salts and EDTA, although all activity is released to the supernatant in the presence of Triton-X-100 or under hypotonic conditions. The properties of bound enzyme are similar to those of free enzyme in cell extracts. It is proposed that osmotically sensitive plasma membrane vesicles, containing cytoplasm fragments, are formed during homogenization. Low malic enzyme activities are also associated with Cucurbita proplastids.