Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

BACKGROUND: Allergic reactions to ß-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. METHODS: We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. RESULTS: We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. CONCLUSIONS: These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response.

Hypersensitivity reactions to ß-lactams: relevance of hapten-protein conjugates.

ß-Lactams (BL) are the drugs most frequently involved in allergic reactions. They are classified according to their chemical structure as penicillins, cephalosporins, monobactams, carbapenems, and clavams. All BL antibiotics have a BL ring that is fused to a 5-member or 6-member ring (except in monobactams) and has 1, 2 or 3 side chains (except in clavams). Differences in chemical structure mean that a wide range of BLs are recognized by the immune system, and patients may experience clinical reactions to one BL while tolerating others. Diagnosis is based on skin and in vitro testing, although both display low sensitivity, possibly because they are based on drugs or drug conjugates that are not optimally recognized by the immune system. BLs are haptens that need to bind to proteins covalently to elicit an immune response. These drugs have a high capacity to form covalent adducts with proteins through nucleophilic attack of amino groups in proteins on the BL ring. Allergenic determinants have been described for all BLs, although benzylpenicillin is the most widely studied. Moreover, formation of BL-protein adducts is selective, as we recently demonstrated for amoxicillin, which mainly modifies albumin, transferrin, and immunoglobulin heavy and light chains in human serum. Given the complexity of BL allergy, understanding the immunological mechanisms involved and optimization of diagnostic methods require multidisciplinary approaches that take into account the chemical structures of the drugs and the carrier molecules, as well as the patient immune response.

Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells.

Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the L-arginine-nitric oxide-cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 micromol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 micromol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.

Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis.

IL-2 deprivation triggers apoptosis in the murine T cell line TS1alphabeta, a process that can be blocked by overexpression of Bcl-2. Here we show that Bcl-2 and Ras proteins interact in mitochondria from TS1alphabeta cells in the presence or absence of IL-2, as evidenced by co-immunoprecipitation. All three Ras proteins, K-, N- and H-Ras, interact with Bcl-2; however, their mitochondrial localization is differentially regulated in IL-2-supplemented or -deprived cells. K-Ras is found in mitochondria only in IL-2-supplemented cells, whereas H-Ras is observed in mitochondria only after IL-2 withdrawal. N-Ras is detected in mitochondria under both experimental conditions. Bcl-2 transfection partially restored K- and N-Ras association with mitochondria in IL-2-deprived cells and rendered H-Ras association independent of IL-2 withdrawal. Inhibitors of Ras posttranslational processing did not alter the IL-2-induced differential pattern of mitochondrial localization. The processed forms of K- and N-Ras associated with mitochondria, although unprocessed H-Ras was also detected in mitochondria from mevastatin-treated cells. These results evidence a distinct behavior among the three Ras proteins in TS1alphabeta cells, depending on IL-2 supply, and suggest homologue-specific roles for Ras proteins in IL-2-dependent events.

Novel aspects of Ras proteins biology: regulation and implications.

The importance of Ras proteins as crucial crossroads in cellular signaling pathways has been well established. In spite of the elucidation of the mechanism of RAS activation by growth factors and the delineation of MAP kinase cascades, the overall framework of Ras interactions is far from being complete. Novel regulators of Ras GDP/GTP exchange have been identified that may mediate the activation of Ras in response to changes in intracellular calcium and diacylglycerol. The direct activation of Ras by free radicals such as nitric oxide also suggests potential regulation of Ras function by the cellular redox state. In addition, the array of Ras effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a dual regulator of cellular functions, playing either positive or negative roles in the regulation of proliferation and apoptosis. The signals transmitted by Ras may be modulated by other pathways triggered in parallel, resulting in the final order for proliferation or apoptosis. The diversity of ras-mediated effects may be related in part to differential involvement of Ras homologues in distinct cellular processes. The study of Ras posttranslational modifications has yielded a broad battery of inhibitors that have been envisaged as anti-cancer agents. Although an irreversible modification, Ras isoprenylation appears to be modulated by growth factors and by the activity of the isoprenoid biosynthetic pathway, which may lead to changes in Ras activity.

The stereochemical requirement for protein kinase C activation by 3-methyldiglycerides matches that found in naturally occurring tumor promoters aplysiatoxins.

Protein kinase C is stereospecifically activated by sn-1,2-(S)-diglycerides. A second chiral center was introduced into the diglycerides by preparing the 3-methyl derivatives. The activation of protein kinase C was also stereospecific with respect to the new chiral center established at the C3 position of the methylated diglycerides. The stereospecifically of protein kinase C directed towards the C2 and C3 positions of the diglycerides is matched in the analogous C29 and C30 stereocenters of the tumor promoting debromoaplysiatoxins. This finding strengthens the view that the structurally diverse tumor promotors contain the embedded diglyceride-like pharmacophore.

Localization of rap1 and rap2 proteins in the gelatinase-containing granules of human neutrophils.

The subcellular localization of rap proteins in resting human neutrophils was investigated by immunoblot analysis with specific anti-rap2 and anti-rap1 antibodies of the membrane proteins obtained from distinct subcellular fractions. Rap2 protein was mainly located in gelatinase-containing granules, whereas rap1 protein was detected both in gelatinase-containing granules and in fractions enriched in plasma membrane. Neither rap1 nor rap2 proteins were found in the cytosol or in azurophilic granules. Rap2B, not rap2A, appeared to be the major rap2 protein in human neutrophils. The identification and subcellular localization of rap1 and rap2 proteins at the membranes of gelatinase-rich granules suggest that these proteins could play a role in the regulation of the rapid and selective mobilization of gelatinase-containing granules in human neutrophils.

Analogs of farnesylcysteine induce apoptosis in HL-60 cells.

S-Farnesyl-thioacetic acid (FTA), a competitive inhibitor of isoprenylated protein methyltransferase, potently suppressed the growth of HL-60 cells and induced apoptosis, as evidenced by the development of increased annexin-V binding, decreased binding of DNA dyes and internucleosomal DNA degradation. FTA did not impair the membrane association of ras proteins, conversely, it brought about a decrease in the proportion of ras present in the cytosolic fraction. Farnesylated molecules which are weak inhibitors of the methyltransferase also induced DNA laddering and reduced the proportion of cytosolic ras. These findings suggest that neither inhibition of isoprenylated protein methylation nor impairment of ras membrane association are essential for apoptosis induced by farnesylcysteine analogs.

Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide.

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.

Regulation of cyclooxygenase-2 expression by nitric oxide in cells.

Two different cyclooxygenases (COXs) are functional in mammals: COX-1 and COX-2. COX-2 is mainly an inducible isoform that shares significant features with inducible nitric oxide synthase (iNOS) in terms of its tissue distribution and participation in pathophysiological phenomena. Furthermore, the product of iNOS catalysis, nitric oxide (NO), is an important regulator of COX-2 activity and expression, and the products of COX-1 and COX-2 (diverse prostanoids) may also influence iNOS expression. Both positive and negative effects of NO on COX-2 expression have been encountered in experimental systems, showing that the outcome of the NO-COX-2 interaction is exquisitely dependent upon the temporal frame and the cell type studied. The pathophysiological significance of NO-COX cross-talk also arises from in vivo studies, in which most evidence points to a positive effect of NO on COX-2 activity and/or expression. This emphasizes the need to understand the underlying mechanisms. Among these, the capacity of NO and its effector cyclic GMP to modulate the function of several target proteins, including transcription factors such as nuclear factor-kappaB and activator protein-1, appears as the key pathway by which NO may regulate COX-2 expression. Given the capacity of some prostanoids to modulate the inflammatory response, the interplay between NO synthase and COX pathways stands at the center of the pathophysiological basis of inflammatory diseases.

The interaction of cycloserine with pyruvate and other biologically relevant alpha-ketoacids.

The ability of cycloserine solutions to deplete alpha-oxoacids has been found to be correlated with the spontaneous transformation of cycloserine into a derivative dimer (2,5-bis-(aminoxymethyl)-3,6-diketopiperazine). Synthetic dimer was found to react rapidly with pyruvate to form the expected oxime. Two lines of evidence indicate that it is the cycloserine dimer and not cycloserine itself that reacts with alpha-ketoacid. First, the 1H NMR spectrum of the purified oxime is superimposable with that arising when the dimer and pyruvate are mixed and the spectrum taken immediately thereafter. Second, the mass spectrum of the reaction product of cycloserine dimer and methylpyruvate is totally consistent with the formation of a stable oxime derivative. Furthermore, when cycloserine is incubated with pyruvate the oxime derived from the dimer is found. These observations clearly indicate that cycloserine in solution can have chemical activities in addition to its ability to interfere with pyridoxal dependent reactions. On these grounds it is concluded that any biological action of cycloserine should be interpreted cautiously.

Early effects of copper accumulation on methionine metabolism.

Wilson's disease is characterized by longterm hepatic accumulation of copper leading to liver disease with reduction of S-adenosylmethionine synthesis. However, the initial changes in this pathway remain unknown and constitute the objective of the present study. Using the Long Evans Cinnamon rat model, early alterations were detected in the mRNA and protein levels, as well as in the activities of several enzymes of the methionine cycle. Notably, the main change was a redox-mediated 80% decrease in the mRNA levels of the methionine adenosyltransferase regulatory subunit as compared to the control group. Moreover, changes in S-adenosylmethionine, S-adenosylhomocysteine, methionine and glutathione levels were also observed. In addition, in vitro experiments show that copper affects the activity and folding of methionine adenosyltransferase catalytic subunits. Taken together, these observations indicate that early copper accumulation alters methionine metabolism with a pattern distinct from that described previously for other liver diseases.

Betaine homocysteine S-methyltransferase: just a regulator of homocysteine metabolism?

Betaine homocysteine methyltransferase (BHMT), a Zn(2+)-dependent thiolmethyltransferase, contributes to the regulation of homocysteine levels, increases in which are considered a risk factor for cardiovascular diseases. Most plasma homocysteine is generated through the liver methionine cycle, in which BHMT metabolizes approximately 25% of this non-protein amino acid. This process allows recovery of one of the three methylation equivalents used in phosphatidylcholine synthesis through transmethylation, a major homocysteine-producing pathway. Although BHMT has been known for over 40 years, the difficulties encountered in its isolation precluded detailed studies until very recently. Thus, the last 10 years, since the sequence became available, have yielded extensive structural and functional data. Moreover, recent findings offer clues for potential new functions for BHMT. The purpose of this review is to provide an integrated view of the knowledge available on BHMT, and to analyze its putative roles in other processes through interactions uncover to date.

Inhibition of isoprenoid biosynthesis induces apoptosis in human promyelocytic HL-60 cells.

Protein isoprenylation is a posttranslational modification that facilitates membrane association and biological activity of a number of proteins. Mevalonate is the precursor of cellular sterols as well as of isoprenoid lipids involved in protein modification. In this study we show that HL-60 cells treated with lovastatin, an inhibitor of mevalonate synthesis, exhibit alterations in growth and morphology, as well as changes in the subcellular distribution of isoprenylated proteins like nuclear lamin A and p21Ras. Moreover, they are induced to die via apoptosis, as evidenced by the appearance of a typical DNA fragmentation pattern. Lovastatin-induced DNA fragmentation can be specifically prevented by mevalonate. The failure of several products of the mevalonate pathway, including cholesterol, to overcome lovastatin effect, points to the involvement of isoprenylated proteins in the mechanisms suppressing cell death.

Inhibition of N-linked glycosylation induces early apoptosis in human promyelocytic HL-60 cells.

Inhibition of protein N-glycosylation by tunicamycin induced morphological changes characteristic of apoptosis in human promyelocytic HL-60 cells. Internucleosomal DNA fragmentation could be detected after short-time incubation (between 6 and 9 h) of HL-60 cells with low doses of tunicamycin (0.05 micrograms/ml). Under these conditions the synthesis of glycoproteins was reduced to 17% of control values, while no significant changes in the rates of total protein synthesis could be observed. Tunicamycin ability to induce DNA fragmentation was in good correlation with its potency as glycosylation inhibitor in several myeloid cell lines. Tunicamycin-induced apoptosis was potentiated by activation of protein kinease C (PKC) by phorbol esters and partially prevented by the PKC inhibitor staurosporine. Inhibitors of RNA and protein synthesis displayed a protective effect. Treatment of HL-60 cells with tunicamycin did not elicit the expression of cell surface differentiation antigens or their ability to generate superoxide anion. In contrast, tunicamycin significantly inhibited these processes during dimethyl sulfoxide (DMSO)-induced myeloid differentiation. These observations indicate that the main effect of tunicamycin in HL-60 cells is the induction of apoptosis.

Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease.

Protein isoprenylation is a post-translational modification essential for the biological activity of G-proteins. Inhibition of protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60 cells, a process of active cell death characterized by the internucleosomal degradation of genomic DNA. In this article we show that LOV-induced apoptosis is associated with intracellular acidification and that activation of the Na+/H+ antiporter induces a raise in pHi which is sufficient to prevent or arrest DNA digestion. First, LOV induced a decrease in pHi which was dose-dependent and correlated with the extent of DNA degradation. Flow cytometry analysis revealed that this acidification was due to the appearance of a subpopulation of cells whose pHi was 0.9 pH units below control values. Cell sorting experiments demonstrated that DNA degradation had occurred only in those cells which had suffered intracellular acidification. LOV-induced apoptosis could be suppressed by mevalonate supplementation, inhibition of protein synthesis, and protein kinase C activation by phorbol myristate acetate. In all three cases, intracellular acidification was abolished. Inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride induced DNA degradation in HL-60 cells per se and suppressed the protective effect of phorbol myristate acetate. LOV-induced intracellular acidification was not due to a complete inhibition of the Na+/H+ antiporter. In fact, LOV-treated cells were able to respond to phorbol myristate acetate stimulation of the Na+/H+ antiporter with a marked increase in pHi. This effect was accompanied by a rapid arrest of DNA digestion. These observations illustrate the strong pH dependence of LOV-induced DNA degradation, thus providing a connection between the activation of the Na+/H+ antiporter and the suppression of apoptosis.

Key role of L-alanine in the control of hepatic protein synthesis.

We investigated the effects of administration of single amino acids to starved rats on the regulation of protein synthesis in the liver. Of all the amino acids tested, only alanine, ornithine and proline promoted statistically significant increases in the extent of hepatic polyribosome aggregation. The most effective of these was alanine, whose effect of promoting polyribosomal aggregation was accompanied by a decrease in the polypeptide-chain elongation time. The following observations indicate that alanine plays an important physiological role in the regulation of hepatic protein synthesis. Alanine was the amino acid showing the largest decrease in hepatic content in the transition from high (fed) to low (starved) rates of protein synthesis. The administration of glucose or pyruvate is also effective in increasing liver protein synthesis in starved rats, and their effects were accompanied by an increased hepatic alanine content. An increase in hepatic ornithine content does not lead to an increased protein synthesis, unless it is accompanied by an increase of alanine. The effect of alanine is observed either in vivo, in rats pretreated with cycloserine to prevent its transamination, or in isolated liver cells under conditions in which its metabolic transformation is fully impeded.