Characterization of TseB: A new actor in cell wall elongation in Bacillus subtilis.

Penicillin-binding proteins (PBPs) are crucial enzymes of peptidoglycan assembly and targets of ß-lactam antibiotics. However, little is known about their regulation. Recently, membrane proteins were shown to regulate the bifunctional transpeptidases/glycosyltransferases aPBPs in some bacteria. However, up to now, regulators of monofunctional transpeptidases bPBPs have yet to be revealed. Here, we propose that TseB could be such a PBP regulator. This membrane protein was previously found to suppress tetracycline sensitivity of a Bacillus subtilis strain deleted for ezrA, a gene encoding a regulator of septation ring formation. In this study, we show that TseB is required for B. subtilis normal cell shape, tseB mutant cells being shorter and wider than wild-type cells. We observed that TseB interacts with PBP2A, a monofunctional transpeptidase. While TseB is not required for PBP2A activity, stability, and localization, we show that the overproduction of PBP2A is deleterious in the absence of TseB. In addition, we showed that TseB is necessary not only for efficient cell wall elongation during exponential phase but also during spore outgrowth, as it was also observed for PBP2A. Altogether, our results suggest that TseB is a new member of the elongasome that regulates PBP2A function during cell elongation and spore germination.

Epigenetic down-regulation of the HIST1 locus predicts better prognosis in acute myeloid leukemia with NPM1 mutation.

BACKGROUND: The epigenetic machinery is frequently altered in acute myeloid leukemia. Focusing on cytogenetically normal (CN) AML, we previously described an abnormal H3K27me3 enrichment covering 70 kb on the HIST1 cluster (6.p22) in CN-AML patient blasts. Here, we further investigate the molecular, functional, and prognosis significance of this epigenetic alteration named H3K27me3 HIST1 in NPM1-mutated (NPM1mut) CN-AML. RESULTS: We found that three quarter of the NPM1mut CN-AML patients were H3K27me3 HIST1high. H3K27me3 HIST1high group of patients was associated with a favorable outcome independently of known molecular risk factors. In gene expression profiling, the H3K27me3 HIST1high mark was associated with lower expression of the histone genes HIST1H1D, HIST1H2BG, HIST1H2AE, and HIST1H3F and an upregulation of genes involved in myelomonocytic differentiation. Mass spectrometry analyses confirmed that the linker histone protein H1d, but not the other histone H1 subtypes, was downregulated in the H3K27me3 HIST1high group of patients. H1d knockdown primed ATRA-mediated differentiation of OCI-AML3 and U937 AML cell lines, as assessed on CD11b/CD11c markers, morphological and gene expression analyses. CONCLUSIONS: Our data suggest that NPM1mut AML prognosis depends on the epigenetic silencing of the HIST1 cluster and that, among the H3K27me3 silenced histone genes, HIST1H1D plays a role in AML blast differentiation.

Differential modification of the C. elegans proteome in response to acute and chronic gamma radiation: Link with reproduction decline.

Emission of ionizing radiation (IR) in the environment is a natural phenomenon which can be enhanced by human activities. Ecosystems are then chronically exposed to IR. But environmental risk assessment of chronic exposure suffers from a lack of knowledge. Extrapolation of data from acute to chronic exposure is not always relevant, and can lead to uncertainties as effects could be different between the two irradiation modes, especially regarding reproduction endpoint, which is an ecologically relevant parameter. In the present study, we decided to refine the understanding of the molecular mechanisms involved in response to acute and chronic γ-irradiation by a global proteome label free LC-MS/MS analysis. C. elegans were exposed to 3 common cumulated radiation doses for acute or chronic exposure condition and global modification of the proteome was studied. This analysis of protein expression has demonstrated the modulation of proteins involved in regulatory biological processes such as lipid transport, DNA replication, germ cell development, apoptosis, ion transport, cuticle development, and aging at lower doses than those for which individual effects on reproduction have been previously observed. Thus, these proteins could constitute early and sensitive markers of radio-induced reprotoxicity; more specifically HAT-1, RPS-19 in acute and VIT-3 for chronic conditions that are expressed in a dose-dependent manner. Finally, to focus on reproduction process, this analysis showed either repression or overexpression of 12 common proteins in organisms exposed to acute or chronic irradiation, respectively. These proteins include the vitellogenin cluster notably involved in lipid transport and oocyte maturation and proteins involved in cuticle development and molting i.e. COL-14, GLF-1, NOAH-1, NOAH-2, ACN-1. These results show that protein expression modulation is a sensitive and predictive marker of radio-induced reproductive effects, but also highlight limitation of data extrapolation from acute to chronic exposure for environmental risk assessment.

Development of parallel reaction monitoring (PRM)-based quantitative proteomics applied to HER2-Positive breast cancer.

INTRODUCTION: treatments targeting the Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) have improved the natural history of HER2-positive breast cancer. However, except HER2 protein expression and gene amplification, there is no predictive biomarker to guide the HER2-targeted therapies. We developed Parallel reaction monitoring (PRM) a powerful approach, to quantify and evaluate key proteins involved in the HER2 pathway and/or anti-HER2 treatment sensitivity. RESULTS: in BCLs, PRM measurements correlated with western blot immunocytochemistry and transcriptomic data. At baseline, higher expression of HER2, EGFR, PTEN and HER3 but lower expression of phospho-HER2 correlated with trastuzumab sensitivity. Under trastuzumab, PRM demonstrated a decrease in HER2 and an increase in phospho-HER2, which correlated with drug sensitivity. The opposite was observed under lapatinib. HER2 quantification was also correlated with immunohistochemistry in PDXs and clinical breast cancer samples. DISCUSSION: in conclusion, PRM-based assay, developed to quantify proteins of the HER2 pathway in breast cancer samples revealed a large magnitude of expression, which may have relevance in terms of treatment sensitivity. MATERIALS AND METHODS: we first evaluated PRM in term of sensitivity, linearity and reproducibility. PRM was then applied to breast cancer cell lines (BCLs) including BCLs exposed to anti-HER2 agents, patient-derived xenografts (PDXs) and frozen breast cancer samples.

Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis.

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.

Responses of the marine diatom Thalassiosira pseudonana to changes in CO2 concentration: a proteomic approach.

The concentration of CO2 in many aquatic systems is variable, often lower than the KM of the primary carboxylating enzyme Rubisco, and in order to photosynthesize efficiently, many algae operate a facultative CO2 concentrating mechanism (CCM). Here we measured the responses of a marine diatom, Thalassiosira pseudonana, to high and low concentrations of CO2 at the level of transcripts, proteins and enzyme activity. Low CO2 caused many metabolic pathways to be remodeled. Carbon acquisition enzymes, primarily carbonic anhydrase, stress, degradation and signaling proteins were more abundant while proteins associated with nitrogen metabolism, energy production and chaperones were less abundant. A protein with similarities to the Ca2+/ calmodulin dependent protein kinase II_association domain, having a chloroplast targeting sequence, was only present at low CO2. This protein might be a specific response to CO2 limitation since a previous study showed that other stresses caused its reduction. The protein sequence was found in other marine diatoms and may play an important role in their response to low CO2 concentration.

Kinetic analysis of mouse brain proteome alterations following Chikungunya virus infection before and after appearance of clinical symptoms.

Recent outbreaks of Chikungunya virus (CHIKV) infection have been characterized by an increasing number of severe cases with atypical manifestations including neurological complications. In parallel, the risk map of CHIKV outbreaks has expanded because of improved vector competence. These features make CHIKV infection a major public health concern that requires a better understanding of the underlying physiopathological processes for the development of antiviral strategies to protect individuals from severe disease. To decipher the mechanisms of CHIKV infection in the nervous system, a kinetic analysis on the host proteome modifications in the brain of CHIKV-infected mice sampled before and after the onset of clinical symptoms was performed. The combination of 2D-DIGE and iTRAQ proteomic approaches, followed by mass spectrometry protein identification revealed 177 significantly differentially expressed proteins. This kinetic analysis revealed a dramatic down-regulation of proteins before the appearance of the clinical symptoms followed by the increased expression of most of these proteins in the acute symptomatic phase. Bioinformatic analyses of the protein datasets enabled the identification of the major biological processes that were altered during the time course of CHIKV infection, such as integrin signaling and cytoskeleton dynamics, endosome machinery and receptor recycling related to virus transport and synapse function, regulation of gene expression, and the ubiquitin-proteasome pathway. These results reveal the putative mechanisms associated with severe CHIKV infection-mediated neurological disease and highlight the potential markers or targets that can be used to develop diagnostic and/or antiviral tools.

Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D) and meta involving a catechol 2,3 dioxygenase (C23D). Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively) and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1)) suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

The first genomic and proteomic characterization of a deep-sea sulfate reducer: insights into the piezophilic lifestyle of Desulfovibrio piezophilus.

Desulfovibrio piezophilus strain C1TLV30(T) is a piezophilic anaerobe that was isolated from wood falls in the Mediterranean deep-sea. D. piezophilus represents a unique model for studying the adaptation of sulfate-reducing bacteria to hydrostatic pressure. Here, we report the 3.6 Mbp genome sequence of this piezophilic bacterium. An analysis of the genome revealed the presence of seven genomic islands as well as gene clusters that are most likely linked to life at a high hydrostatic pressure. Comparative genomics and differential proteomics identified the transport of solutes and amino acids as well as amino acid metabolism as major cellular processes for the adaptation of this bacterium to hydrostatic pressure. In addition, the proteome profiles showed that the abundance of key enzymes that are involved in sulfate reduction was dependent on hydrostatic pressure. A comparative analysis of orthologs from the non-piezophilic marine bacterium D. salexigens and D. piezophilus identified aspartic acid, glutamic acid, lysine, asparagine, serine and tyrosine as the amino acids preferentially replaced by arginine, histidine, alanine and threonine in the piezophilic strain. This work reveals the adaptation strategies developed by a sulfate reducer to a deep-sea lifestyle.

Identification of salivary antigenic markers discriminating host exposition between two European ticks: Rhipicephalus sanguineus and Dermacentor reticulatus.

To succeed blood meal, ticks inject salivary proteins to mammalian hosts, eliciting an antibody response against these foreign antigens. Although this immune response has been proposed as a surrogate marker of exposure to tick bites, identification of the corresponding antigens remains elusive. For this aim, a comparison by immunoblots of the kinetic IgG responses to protein salivary gland extracts from two European tick species, Rhipicephalus sanguineus or Dermacentor reticulatus, in rabbits was performed. A singularity in the immune patterns was observed according to rabbit exposure status and depending on the antigen source. Six and five bands were found specifically associated to R. sanguineus and to D. reticulatus exposures, respectively. The identity of these salivary antigenic proteins was determined using an original immunoproteomic approach. The utilization of these tick salivary proteins as biomarker candidates to discriminate R. sanguineus and/or D. reticulatus tick exposure or to develop anti-tick vaccines is discussed.

A UPR-independent infection-specific role for a BiP/GRP78 protein in the control of antimicrobial peptide expression in C. elegans epidermis.

The nematode C. elegans responds to infection by the fungus Drechmeria coniospora with a rapid increase in the expression of antimicrobial peptide genes. To investigate further the molecular basis of this innate immune response, we took a two-dimensional difference in-gel electrophoresis (2D-DIGE) approach to characterize the changes in host protein that accompany infection. We identified a total of 68 proteins from differentially represented spots and their corresponding genes. Through class testing, we identified functional categories that were enriched in our proteomic data set. One of these was "protein processing in endoplasmic reticulum," pointing to a potential link between innate immunity and endoplasmic reticulum function. This class included HSP-3, a chaperone of the BiP/GRP78 family known to act coordinately in the endoplasmic reticulum with its paralog HSP-4 to regulate the unfolded protein response (UPR). Other studies have shown that infection of C. elegans can provoke a UPR. We observed, however, that in adult C. elegans infection with D. coniospora did not induce a UPR, and conversely, triggering a UPR did not lead to an increase in expression of the well-characterized antimicrobial peptide gene nlp-29. On the other hand, we demonstrated a specific role for hsp-3 in the regulation of nlp-29 after infection that is not shared with hsp-4. Epistasis analysis allowed us to place hsp-3 genetically between the Tribbles-like kinase gene nipi-3 and the protein kinase C delta gene tpa-1. The precise function of hsp-3 has yet to be determined, but these results uncover a hitherto unsuspected link between a BiP/GRP78 family protein and innate immune signaling.

Global response of Plasmodium falciparum to hyperoxia: a combined transcriptomic and proteomic approach.

BACKGROUND: Over its life cycle, the Plasmodium falciparum parasite is exposed to different environmental conditions, particularly to variations in O2 pressure. For example, the parasite circulates in human venous blood at 5% O2 pressure and in arterial blood, particularly in the lungs, at 13% O2 pressure. Moreover, the parasite is exposed to 21% O2 levels in the salivary glands of mosquitoes. METHODS: To study the metabolic adaptation of P. falciparum to different oxygen pressures during the intraerythrocytic cycle, a combined approach using transcriptomic and proteomic techniques was undertaken. RESULTS: Even though hyperoxia lengthens the parasitic cycle, significant transcriptional changes were detected in hyperoxic conditions in the late-ring stage. Using PS 6.0 ™ software (Ariadne Genomics) for microarray analysis, this study demonstrate up-expression of genes involved in antioxidant systems and down-expression of genes involved in the digestive vacuole metabolism and the glycolysis in favour of mitochondrial respiration. Proteomic analysis revealed increased levels of heat shock proteins, and decreased levels of glycolytic enzymes. Some of this regulation reflected post-transcriptional modifications during the hyperoxia response. CONCLUSIONS: These results seem to indicate that hyperoxia activates antioxidant defence systems in parasites to preserve the integrity of its cellular structures. Moreover, environmental constraints seem to induce an energetic metabolism adaptation of P. falciparum. This study provides a better understanding of the adaptive capabilities of P. falciparum to environmental changes and may lead to the development of novel therapeutic targets.

Proteomic analysis revealed alterations of the Plasmodium falciparum metabolism following salicylhydroxamic acid exposure.

OBJECTIVES: Although human respiratory metabolism is characterized by the mitochondrial electron transport chain, some organisms present a "branched respiratory chain." This branched pathway includes both a classical and an alternative respiratory chain. The latter involves an alternative oxidase. Though the Plasmodium falciparum alternative oxidase is not yet identified, a specific inhibitor of this enzyme, salicylhydroxamic acid (SHAM), showed a drug effect on P. falciparum respiratory function using oxygen consumption measurements. The present study aimed to highlight the metabolic pathways that are affected in P. falciparum following SHAM exposure. DESIGN: A proteomic approach was used to analyze the P. falciparum proteome and determine the metabolic pathways altered following SHAM treatment. To evaluate the SHAM effect on parasite growth, the phenotypic alterations of P. falciparum after SHAM or/and hyperoxia exposure were observed. RESULTS: After SHAM exposure, 26 proteins were significantly deregulated using a fluorescent two dimensional-differential gel electrophoresis. Among these deregulated proteins, some were particularly involved in energetic metabolism. And the combinatory effect of SHAM/hyperoxia seems deleterious for the growth of P. falciparum. CONCLUSION: Our results indicated that SHAM appears to activate glycolysis and decrease stress defense systems. These data provide a better understanding of parasite biology.

Specific antibody responses against membrane proteins of erythrocytes infected by Plasmodium falciparum of individuals briefly exposed to malaria.

BACKGROUND: Plasmodium falciparum infections could lead to severe malaria, principally in non-immune individuals as children and travellers from countries exempted of malaria. Severe malaria is often associated with the sequestration of P. falciparum-infected erythrocytes in deep micro-vascular beds via interactions between host endothelial receptors and parasite ligands expressed on the surface of the infected erythrocyte. Although, serological responses from individuals living in endemic areas against proteins expressed at surface of the infected erythrocyte have been largely studied, seldom data are available about the specific targets of antibody response from travellers. METHODS: In order to characterize antigens recognized by traveller sera, a comparison of IgG immune response against membrane protein extracts from uninfected and P. falciparum-infected red blood cells (iRBC), using immunoblots, was performed between non exposed individuals (n = 31) and briefly exposed individuals (BEI) (n = 38) to malaria transmission. RESULTS: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group. Some of these antigenic proteins were identified by an original immuno-proteomic approach. CONCLUSION: Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.

The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the "Brucella-containing vacuole" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

Antifungal innate immunity in C. elegans: PKCdelta links G protein signaling and a conserved p38 MAPK cascade.

Like other multicellular organisms, the model nematode C. elegans responds to infection by inducing the expression of defense genes. Among the genes upregulated in response to a natural fungal pathogen is nlp-29, encoding an antimicrobial peptide. In a screen for mutants that fail to express nlp-29 following fungal infection, we isolated alleles of tpa-1, homologous to the mammalian protein kinase C (PKC) delta. Through epistasis analyses, we demonstrate that C. elegans PKC acts through the p38 MAPK pathway to regulate nlp-29. This involves G protein signaling and specific C-type phospholipases acting upstream of PKCdelta. Unexpectedly and unlike in mammals, tpa-1 does not act via D-type protein kinases, but another C. elegans PKC gene, pkc-3, functions nonredundantly with tpa-1 to control nlp-29 expression. Finally, the tribbles-like kinase nipi-3 acts upstream of PKCdelta in this antifungal immune signaling cascade. These findings greatly expand our understanding of the pathways involved in C. elegans innate immunity.

ABCA2 is a marker of neural progenitors and neuronal subsets in the adult rodent brain.

The notion that the ATP-binding cassette transporter-A2 (ABCA2) may be involved in brain sterol homeostasis and is associated with early onset Alzheimer's disease led us to explore its neural expression. Our data support and extend the previous reports on ABCA2 expression by oligodendrocytes. They evidence that ABCA2 (i) is located in intracellular vesicles, identified in transfected cells as lysosome-related organelles only partially overlapping with classical endolysosomes; (ii) is a marker of neural progenitors as it is expressed in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampal formation, sites of continual neurogenesis in the adult brain, and in nestin(+) cells differentiated in vitro from embryonic stem cells; (iii) persists, in the adult rodent brain, in a subset of GABAergic and glutamatergic neurons. Considering that the latter are targets of Alzheimer's lesions, these data provide a new rationale to explore the neuropathological consequences of ABCA2 functional dysregulations.

Cooperation between engulfment receptors: the case of ABCA1 and MEGF10.

The engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway.

A novel reporter strain to follow Cre-mediated recombination in T and NK cells.

The Cre-loxP system permits the generation of mouse models in which the fate of a cell can be followed through time. Such approach is of great value in immunology because it may allow lineage studies and the dissection of the contribution of specific effector T cells to long-term memory responses or autoimmune responses. An essential component of such a strategy is the development of appropriate reporter strains of mice in which the inducible reporter molecule is not immunogenic and is well expressed at the cell surface of T cells. We describe here a novel reporter strain of mice that is designed to fulfill these criteria and show that this strain permits the monitoring of Cre-mediated recombination in both T cells and NK cells.

Deletion of CCK2 receptor in mice results in an upregulation of the endogenous opioid system.

Stimulation of the brain CCK2 receptor by the C-terminal octapeptide CCK8 of cholecystokinin (CCK) negatively modulates opioid responses. This suggests the existence of physiologically relevant interactions between endogenous CCK and opioid peptides, opening new perspectives particularly in the treatment of pain or drug addiction. CCK2 receptor-deficient mice were used to analyze the incidence of this gene invalidation on opioid system. Compared with wild-type mice, mutants exhibited the following: (1) a hypersensitivity to the locomotor activity induced by inhibitors of enkephalin catabolism or by morphine; (2) a spontaneous hyperalgesia to thermal nociceptive stimulus, which was reversed by previous administration of the NMDA antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], and a large reduction in analgesic effects of endogenous or exogenous opioids; and (3) a more severe withdrawal syndrome after chronic morphine treatment. As expected, stimulation of mu, delta, and D2 receptors on brain tissue of wild-type animals induced a dose-dependent decrease in adenylate cyclase activity, whereas a striking mirror effect was observed in mutants. All of these results suggest that the absence, in knock-out mice, of the negative feedback control on the opioid system, normally performed out by CCK2 receptor stimulation, results in an upregulation of this system. These biochemical and pharmacological results demonstrate the critical role played by CCK2 receptors in opioid-dependent responses.