An engineered PET depolymerase to break down and recycle plastic bottles.

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.

In a high-dose melphalan setting, palifermin compared with placebo had no effect on oral mucositis or related patient's burden.

This randomized-controlled trial studied the efficacy of palifermin in a chemotherapy-only, high-dose Melphalan (HDM) transplant setting, to reduce oral mucositis (OM) and its sequelae measured by patient-reported outcomes (PRO) and medical resource use. Palifermin, relative to placebo was given either pre-/post-HDM or pre-HDM in patients with multiple myeloma (MM) undergoing auto-SCT at 39 European centers. Oral cavity assessment (WHO) and PRO questionnaires (oral mucositis daily questionnaire (OMDQ) and EQ 5D) were used in 281 patients (mean age 56, ± s.d.=8 years). 57 patients received placebo. One hundred and fifteen subjects were randomized to pre-/post-HDM receiving palifermin on 3 consecutive days before HDM and after auto-SCT and 109 patients were randomized to pre-HDM, receiving palifermin (60 µg/kg/day) i.v. for 3 consecutive days before HDM. There was no statistically significant difference in maximum OM severity. Severe OM occurred in 37% (placebo), 38% (pre-/post-HDM) and 24% (pre-HDM) of patients. No significant difference was observed with respect to PRO assessments or medical resource use, but more infections and fever during neutropenia were reported in pre-/post-HDM vs placebo (for example, 51 and 26%). To conclude, palifermin was unable to reduce OM or OM-related patient's burden in MM transplant patients.

Kinetic and mechanical basis of rolling through an integrin and novel Ca2+-dependent rolling and Mg2+-dependent firm adhesion modalities for the alpha 4 beta 7-MAdCAM-1 interaction.

We studied interactions in shear flow of cells bearing integrins alpha4beta1 or alpha4beta7 with VCAM-1 and MAdCAM-1 substrates in different divalent cations. Interestingly, Ca(2+) was essential for tethering in flow and rolling interactions through both alpha4 integrins. Mg(2+) promoted firm adhesion of alpha4beta7-expressing cells on MAdCAM-1 but with much lower tethering efficiency in shear flow. The k(off) degrees of 1.28 s(-1) and resistance of the receptor-ligand bond to force (estimated as a bond interaction distance or sigma) for transient tethers on MAdCAM-1 were similar to values for E- and P-selectins. By contrast to results in Ca(2+) or Ca(2+) + Mg(2+), in Mg(2+) the alpha4beta7-MAdCAM-1 k(off) degrees decreased 20-fold to 0.046 s(-1), and the bond was weaker, providing an explanation for the finding of firm adhesion under these conditions. Shear enhanced tethering to MAdCAM-1, thereby contributing to the stability of rolling. Comparisons to selectins demonstrate that the kinetic and mechanical properties of the alpha4beta7 integrin are well suited to its intermediate position in adhesion cascades, in which it bridges rapid rolling through selectins to firm adhesion through beta2 integrins.

Human monocytes possess a serine protease activity capable of degrading HIV-1 reverse transcriptase in vitro.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) plays a central role in the virus replication cycle. We found that HIV-1 RT was rapidly degraded when incubated with cell extracts obtained from human peripheral blood cells. The proteolytic activity responsible for the in vitro degradation of RT was present in monocytes and their precursors. Interestingly, this activity was downregulated upon cell activation or differentiation along the macrophage pathway. The proteolytic process appears specific for HIV-1 RT since other HIV-1 proteins were not degraded upon incubation in the same extracts. Although the degradation of RT was unaffected by specific proteasome inhibitors, it could be inhibited by PMSF and aprotinin, suggesting the involvement of a serine protease. Upon cell fractionation, this serine protease was found to be associated with the microsomal fraction and displayed an apparent molecular weight of approximately 2000 kDa, as determined by gel filtration. Our results suggest that a giant serine protease, different from tripeptidyl peptidase II, is involved in the in vitro degradation of HIV-1 RT. The possibility of an in vivo interaction between HIV-1 RT and a cell-type-specific serine protease is discussed.

Sarcoma after radiation therapy: retrospective multiinstitutional study of 80 histologically confirmed cases. Radiation Therapist and Pathologist Groups of the Fédération Nationale des Centres de Lutte Contre le Cancer.

PURPOSE: To evaluate the best strategy for treatment of sarcoma that occurs after radiation therapy. MATERIALS AND METHODS: Records were retrospectively reviewed for 80 patients with a confirmed histologic diagnosis of sarcoma that occurred after radiation therapy performed during 1975-1995. The patients were treated for breast cancer (n = 33, 42%), non-Hodgkin lymphoma (n = 9, 11%), cervical cancer (n = 9, 11%), benign lesions (n = 4, 5%), or other tumors (n = 25, 31%). Sarcoma occurred after a mean latency of 12 years (range, 3-64 years), with most (70%) developing in the soft tissue. Treatment included surgery (28 patients), surgery and chemotherapy (18 patients), chemotherapy only (15 patients), and radiation therapy (14 patients). RESULTS: By the end of the study, 51 patients were dead, including 46 due to sarcoma. Median survival was 23 months. Overall survival rates at 2 and 5 years, respectively, were 69% and 39% for patients treated with surgery, 10% and 0% for those treated with chemotherapy, and 52% and 35% for those treated with surgery and chemotherapy (P =.001). The 2- and 5-year rates for survival without recurrence were 54% and 32%, respectively. CONCLUSION: The results confirm the beneficial effect of surgery. Further study is needed to explore the roles of combined treatments.

Changes in oxytocin content in rat brain during morphine withdrawal.

In this study the modification in the oxytocin content in different hypothalamic nuclei during morphine withdrawal was analysed. Male rats were implanted with placebo (naïve) or morphine (tolerant/dependent) pellets for 7 days. On day 7, groups of rats received an acute injection of saline s.c. (control) or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. After administration of naloxone to tolerant rats (withdrawal) an increase in the oxytocin content in the paraventricular nucleus (PVN) and median eminence (ME) was found. No changes were found in the arcuate nucleus (AN) and supraoptic nucleus (SON). Present data demonstrate that administration of naloxone to tolerant rats alters the brain oxytocin system, which suggests that this peptide might contribute to the behavioural, emotional and neuroendocrine response to opioid.

Alterations in corticotropin-releasing factor and vasopressin content in rat brain during morphine withdrawal: correlation with hypothalamic noradrenergic activity and pituitary-adrenal response.

The modification in the activity of noradrenergic neurons projecting to the hypothalamus and the pituitary-adrenal response during morphine withdrawal as well its correlation with alterations in corticotropin-releasing factor (CRF) and vasopressin (AVP) content in different brain areas was analyzed. Male rats were implanted with placebo (naïve) or morphine (tolerant/dependent) pellets for 7 days. On day 8, groups of rats received an acute injection of saline s.c. (control) or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. After administration of naloxone to tolerant rats (withdrawal) we found a striking parallelism between an enhanced activity of hypothalamic noradrenergic neurons and an increased corticosterone secretion; concomitantly, the CRF but not the AVP content in the paraventricular nucleus was decreased, which might reflect an increased release of the peptide. During withdrawal, CRF content also was decreased in the arcuate nucleus, whereas no changes were found in the median eminence, dorsomedial, ventromedial nuclei or in the bed nucleus of the stria terminalis. AVP content levels were not modified in arcuate nucleus, supraoptic or in the suprachiasmatic nuclei. Present data suggest that a hypothalamic noradrenergic hypersecretion may be involved in a selectively increased activity of CRF neurons in the paraventricular nucleus and arcuate nucleus and then in the enhanced release of corticosterone induced by morphine withdrawal. However, we did not find any correlation between opioid withdrawal-induced alterations in the pituitary-adrenal axis and AVP modifications.

Differential regulation of corticotropin-releasing factor and vasopressin in discrete brain regions after morphine administration: correlations with hypothalamic noradrenergic activity and pituitary-adrenal response.

The changes in the content of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in discrete brain nuclei during chronic opioids administration have not been well established. We evaluated the effects of acute and chronic morphine administration on the content of CRF and AVP in different hypothalamic and extrahypothalamic (bed nucleus of the stria terminalis, BNST) nuclei in rats. Concomitantly, changes in hypothalamic noradrenaline (NA) turnover [estimated by the 3-methoxy-4-hydroxyphenylethyleneglycol MHPG/NA ratio] and in plasma corticosterone release (as a marker of the activity of the hypothalamus-pituitary-adrenal axis) were determined. Male rats were implanted with placebo (naïve) or morphine (tolerant) pellets for 7 days. On day 8, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg i.p.) and were sacrificed 30 min later. Acute morphine injection to naïve rats increased both the release of corticosterone and the hypothalamic NA turnover. CRF and AVP showed no modifications in the paraventricular nucleus (PVN) or in the median eminence (ME). CRF content decreased in the ventromedian nucleus (VMN) and increased in the BNST, but did not change in the arcuate nucleus (AN). AVP was elevated in the supraoptic nucleus (SON) but not changed in the suprachiasmatic nucleus (SCN). In chronic morphine-treated rats, there was a pronounced decrease in the NA turnover and in the release of corticosterone, which indicates that tolerance develops to the acute effects of morphine. Correspondingly, CRF and AVP were enhanced in the PVN and decreased in the ME, when compared with naïve rats injected with morphine. CRF content was decreased in the AN and in the BNST, but increased in the VMN. The AVP content was decreased in the SON, and no modifications were seen in the SCN. The present study shows that, in addition to the modifications in corticosterone secretion and in hypothalamic NA turnover, chronic morphine administration produces a complex response in the CRF and AVP systems. These modifications might contribute to the behavioral, emotional and neuroendocrine alterations produced during opioid tolerance.

Changes in hypothalamic oxytocin levels during morphine tolerance.

The role of hypothalamic oxytocin neurons in the hypothalamus-pituitary-adrenal (HPA) axis adaptation during opioid tolerance has not been explored. In this study the modification of oxytocin levels in different hypothalamic nuclei was determined after acute or chronic morphine exposure. Male rats were implanted with placebo (naïve) or morphine (tolerant) pellets for 7 days. On day 8, groups of rats received an acute injection of either saline i.p. or morphine (30 mg/kg i.p.) and were sacrificed 30 min later. In morphine-tolerant rats, there was a decrease in the oxytocin content in the median eminence (ME) and in the supraoptic nucleus (SO) after acute injection of saline or morphine. No modifications were seen in the paraventricular nucleus (PVN). The present study demonstrates that chronic morphine administration alters the brain oxytocin system, which suggests that this peptide might contribute to the behavioural, emotional and neuroendocrine responses to opioids.

Prosomes (proteasomes) changes during differentiation are related to the type of inducer.

The core of the 26S proteasome, the 20S prosome, is a highly organized multi-protein complex found in large amount in malignant cells. Differentiation of several cell lines, including the monoblastic U937 and the lymphoblastoid CCRF-CEM, is accompanied by a general decrease in the prosome concentration when phorbol-myrirtic-acetate (PMA) and retinoic acid plus dihydroxyvitamine D3 (RA+VD) are used. Incubation of U937 cells for three days with PMA or RA+VD causes differentiation, but the resulting patterns of prosome labeling in the cell and on the plasma membrane are not the same. In contrast, the same kind of prosome changes occur in U937 and CCRF-CEM cells when PMA is used as inducer. The intracellular distribution of prosomes is also linked to malignancy and differentiation. Prosomes are found in the nucleus and the cytoplasm of cancer cells; and treatment with RA+VD decreases the prosomes in the nucleus whereas PMA causes various prosome proteins changes. These results indicate that prosomes are important in cell regulation and in the expression of malignancy.

Solution structure of the albumin-binding GA module: a versatile bacterial protein domain.

The albumin-binding GA module is found in a family of surface proteins of different bacterial species. It comprises 45 amino acid residues and represents the first known example of contemporary module shuffling. Using 1H NMR spectroscopy we have determined the solution structure of the GA module from protein PAB, a protein of the anaerobic human commensal and pathogen Peptostreptococcus magnus. This structure, the first three-dimensional structure of an albumin-binding protein domain described, was shown to be composed of a left-handed three-helix-bundle. Sequence differences between GA modules with different affinities for albumin indicated that a conserved region in the C-terminal part of the second helix and the flexible sequence between helices 2 and 3 could contribute to the albumin-binding activity. The effect on backbone amide proton exchange rates upon binding to albumin support this assumption. The GA module has a fold that is strikingly similar to the immunoglobulin-binding domains of staphylococcal protein A but it shows no resemblance to the fold shared by the immunoglobulin-binding domains of streptococcal protein G and peptostreptococcal protein L. When the gene sequences, binding properties and thermal stability of these four domains are analysed in relation to their global folds an evolutionary pattern emerges. Thus, in the evolution of novel binding properties mutations are allowed only as long as the energetically favourable global fold is maintained.

The oxidative burst triggered by Salmonella typhimurium in differentiated U937 cells requires complement and a complete bacterial lipopolysaccharide.

The bacterial and serum factors involved in the oxidative response triggered by Salmonella typhimurium in differentiated U937 cells were investigated. Complement activation was shown to be required, using sera deficient in complement factors. An original dot-blot technique was developed to study the activation of complement by either bacteria or purified lipopolysaccharide (LPS). Both O-specific and lipid A segments of LPS were found to play a role in the triggering of the oxidative response. Lipid A was responsible for bacterial C3-derived opsonization by inducing an antibody-independent activation of complement classical pathway, whereas O-specific polysaccharide chains (O-Ag) were involved in cellular activation. Inhibition experiments using anti-cell surface marker monoclonal antibodies showed the involvement of the alpha chain of CR3 (CD11b) in the oxidative response developed by differentiated U937 cells in response to S. typhimurium infection. Whether both iC3b and O-Ag interact with different domains of CR3 or whether the binding of O-Ag occurs via a not yet identified receptor remains to be determined.

Proteasome (prosome) subunit variations during the differentiation of myeloid U937 cells.

20S proteasomes (prosomes/multicatalytic proteinase) are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol-myristate-acetate or retinoic acid plus 1,25-dihydroxy-cholecalciferol by western blot, flow cytometry and immuno-fluoresence. p25K (C3), p27K (IOTA) and p30/33K (C2) subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA + VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno-fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA + VD-induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA-induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non-induced cells; these membrane proteins disappeared when treated with RA + VD, whereas some increased on PMA-induced cells. The differential changes in the distribution and type of proteasomes in RA + VD and PMA-induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

Subcellular distribution and profiles of prosomes (proteasomes-MCP) during differentiation of human lymphoblastic cell line.

The human lymphoblastoid leukemic cell line (CCRF-CEM) was induced to differentiate with phorbol 12-myristate 13-acetate (PMA). During differentiation, assessed by monitoring the cluster of differentiation (CD) profile, the prosome (proteasomes, multi-catalytic proteinase) distribution and composition were studied by microscopy, flow cytometry and Western blot analysis. Changes in prosome subunits were monitored using 3 monoclonal antibodies anti-p23K, p29K and p31K. There were changes in the subcellular distribution of prosome antigens in PMA treated cells compared to untreated cells. The amount of cytoplasmic prosomal antigens decreased during the first three days of differentiation and the membrane antigens increased; meanwhile there was an increase of p53 and no change in actin protein levels. As mitotic cyclins are degraded by the ubiquitin pathway and therefore via the prosome, the decrease observed in differentiated cells suggests that prosomes are involved in the cell cycle and thus in cell proliferation.

Changes in the amount and distribution of prosomal subunits during the differentiation of U937 myeloid cells: high expression of p23K.

Prosomes (Proteasomes/Multicatalytic proteinase (MCP)-complexes) are protein particles built of 28 subunits in variable composition, having proteinase activity. We have studied the changes in prosomal subunits p29K, p31K and the highly expressed p23K during the differentiation of U937 cells. Control cells had little prosomal subunit p31K in the cytoplasm, while p29K antigen was detected in both the nucleus and cytoplasm; more p23K antigen was found in the cytoplasm than in the nucleus. Flow cytometry demonstrated a biphasic intracellular decrease in prosomes during differentiation induced by phorbol-myristic-acetate (PMA) and retinoic acid plus 1,25-dihydroxycholecalciferol (RA + VD). p23K and p29K decreased both in the cytoplasm and the nucleus of differentiated cells, though the p23K antigen was concentrated near vesicles and the plasma membrane in PMA-induced cells. The p31K antigens disappeared from RA + VD-induced cells, while in PMA-induced cells, cytoplasmic labelling was unchanged and nuclear labelling was increased. Small amounts of prosomal proteins p23K and p29K were found on the outer membrane of un-induced cells. While there was no labelling on the outer membrane of RA + VD-induced cells, p23K protein increased on the plasma membrane of PMA-induced cells. The prosome-like particle protein p21K was not present to any significant extent in the intracellular compartment of control or induced cells; however, p21K was detected on the outer surface of control cells and was increased only in PMA-induced cells. The culture medium of control and induced cells contained no p21K, p23K, p29K or p31K. RA + VD seemed to induce a general decrease of prosomal subunits within the cells and at the outer surface, whereas PMA caused a migration toward the plasma membrane and an increase at the outer surface. These changes in the distribution and type of prosomes in RA + VD- and PMA-induced cells indicate that prosomes may play a part in differentiation, especially p23K which is the most highly expressed protein among those studied and presents the more important changes.

Protein PAB, an albumin-binding bacterial surface protein promoting growth and virulence.

The anaerobic bacterium Peptostreptococcus magnus is a human commensal and pathogen. Previous work has shown that strains of P. magnus isolated from patients with gynecological disease (vaginosis) frequently express an immunoglobulin (Ig) light chain-binding protein called protein L. Here we report that strains isolated from localized suppurative infections bind human serum albumin (HSA), whereas commensal isolates bind neither Ig nor HSA. The HSA-binding protein PAB was extracted from the bacterial surface or isolated from the culture supernatant of the P. magnus strain ALB8. Protein PAB was shown to have two homologous HSA-binding domains, GA and uGA. GA is absent in the sequence of a related protein from another P. magnus strain and shows a high degree of homology to the HSA-binding domains of streptococcal protein G. Therefore GA is believed to have recently been shuffled as a module from genes of other bacterial species into the protein PAB gene. This GA module was shown to exhibit a much higher affinity for HSA than uGA and was also found to be present in all of the isolates tested from localized suppurative infections, indicating a role in virulence. Moreover, when peptostreptococci or streptococci expressing the GA module were grown in the presence of HSA, the growth rate was substantially increased. Thus, the HSA binding activity of the GA module adds selective advantages to the bacteria, which increases their virulence in the case of P. magnus strains.

Identification of interdomain sequences promoting the intronless evolution of a bacterial protein family.

In the evolution of eukaryotic genes, introns are believed to have played a major role in increasing the probability of favorable duplication events, chance recombinations, and exon shuffling resulting in functional hybrid proteins. As a rule, prokaryotic genes lack introns, and the examples of prokaryotic introns described do not seem to have contributed to gene evolution by exon shuffling. Still, certain protein families in modern bacteria evolve rapidly by recombination of genes, duplication of functional domains, and as shown for protein PAB of the anaerobic bacterial species Peptostreptococcus magnus, by the shuffling of an albumin-binding protein module from group C and G streptococci. Characterization of a protein PAB-related gene in a P. magnus strain with less albumin-binding activity revealed that the shuffled module was missing. Based on this fact and observations made when comparing gene sequences of this family of bacterial surface proteins interacting with albumin and/or immunoglobulin, a model is presented that can explain how this rapid intronless evolution takes place. A new kind of genetic element is introduced: the recer sequence promoting interdomain, in frame recombination and acting as a structure-less flexibility-promoting spacer in the corresponding protein. The data presented also suggest that antibiotics could represent the selective pressure behind the shuffling of protein modules in P. magnus, a member of the indigenous bacterial flora.

Implication of tyrosine kinases and protein kinase C in dimethyl sulfoxide-induced apoptosis.

We have previously shown that the chemical agent of myeloid differentiation, dimethyl sulfoxide (DMSO), causes apoptosis in human leukemic U937 cells (Château et al. Anal. Cell. Pathol. 1996;10:75-84). Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) led to inhibition of the DMSO-induced apoptosis, suggesting that PKC helps regulate this mechanism by preventing cell death. However, specific inhibitors of PKC (bisindolylmaleimide, D sphingosine), neither triggered apoptosis themselves, nor affected the DMSO-induced apoptosis. Surprisingly, herbimycin A, a potent inhibitor of tyrosine kinases, did not trigger apoptosis itself, but it did prevent DMSO-induced nuclear fragmentation, whereas okadaic acid, an inhibitor of protein phosphatases, triggered apoptosis in U937 cells. These results suggest that DMSO-induced apoptosis requires the activation of an unidentified tyrosine kinase that is probably down-regulated by PKC activation.

Dimethyl sulfoxide-induced apoptosis in human leukemic U937 cells.

Dimethyl sulfoxide (DMSO), which induces differentiation of myeloid cells, was found to cause apoptosis in human leukemic U937 cells. Apoptosis was assessed by DNA electrophoresis and flow cytometry. The time needed to induce apoptosis varied from a few hours to 2-3 days, depending on the concentration of DMSO used. The plasma membrane remained intact long after DNA fragmentation had occurred. DMSO-induced apoptosis was inhibited by zinc ions and, to a lesser extent, by the protein kinase C activator: phorbol 12-myristate 13-acetate (PMA). Cycloheximide and actinomycin D did not prevent DMSO-induced apoptosis, showing that U937 cells do not require protein or RNA synthesis to undergo apoptosis. DMSO induced apoptosis despite the expression of the anti-apoptotic Bcl-2 protein in U937 cells. The amount of Bcl-2 remained unchanged during DMSO-induced apoptosis.

Differentiated U937 cells and human monocytes exhibit a differential production of extracellular oxygen species: O2.- excretion versus H2O2 diffusion.

The nature and the localization of the oxidative response triggered by different stimuli in either differentiated U937 cells and peripheral blood-derived human monocytes was investigated using luminometric and cytofluorometric techniques. Differentiated U937 cells essentially produced extracellular superoxide anion (O2.-), whatever the stimulus used. Monocytes, however, responded to Salmonella typhimurium, phorbol esters, and opsonized zymosan by an intracellular, an extracellular, and both an intra- and extracellular production of oxygen species, respectively. Furthermore, H2O2 but not O2.- was detected in the extracellular oxidative response of monocytes. Using differentiated U937 cells, luminol was found to be as efficient as lucigenin in the detection of extracellular O2.-, providing sufficient concentrations of extracellular horseradish peroxidase were present. However, both azide and histidine inhibited the lucigenin-enhanced chemiluminescence, suggesting an initial and transient production of singlet oxygen by differentiated U937 cells. Taken together these results strongly suggest that, when stimulated, differentiated U937 cells directly excrete O2.- in the extracellular medium while, within monocytes, O2.- is rapidly dismutated in H2O2 which can eventually diffuse outside the cell. Such differences in the oxidative response between the two cell types could be explained by the lack of total closure of the phagosome, only observed in differentiated U937 cells.