Characterization of cardiac-resident progenitor cells expressing high aldehyde dehydrogenase activity.

High aldehyde dehydrogenase (ALDH) activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDH(br)) cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDH(br) cells in the heart has not been evaluated so far. We have characterized ALDH(br) cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDH(br). ALDH(very-br) cells were more frequent in neonatal hearts than adult. ALDH(br) cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDH(very-br) cells, intermediate in ALDH(br) cells, and lowest in ALDH(dim) cells. ALDH1A2 expression was highest in ALDH(very-br) cells, intermediate in ALDH(dim) cells, and lowest in ALDH(br) cells. ALDH1A3 and ALDH2 expression was detectable in ALDH(very-br) and ALDH(br) cells, unlike ALDH(dim) cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDH(br) cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDH(br) cells, unlike ALDH(dim) cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α -actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDH(br) cells declined with cell passage. In conclusion, the cardiac-derived ALDH(br) population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDH(br) cells remains to be evaluated.

Biochemical, morphological and content characterization of synaptobrevin 2-expressing vesicles in astrocytes

Neuron-astrocyte reciprocal communication at synapses has emerged as a novel signalling pathway in brain function. Astrocytes sense the level of synaptic activity and, in turn, influence its efficacy through the regulated release of ''glio- transmitters'' such as glutamate, ATP or D-serine. A calcium- dependent exocytosis is proposed to drive the release of gliotransmitters but its existence is still debated. To shed light onto the mechanisms controlling the storage and the release of gliotransmitters and namely D-serine, we have developed a new method for the immunoisolation of synaptobrevin 2-positive vesicles from rat cortical astrocytes in culture. The purified organelles are clear round shape vesicles of excellent purity as judged by electron microscopy. Immunoblotting analysis revealed that isolated vesicles contain most of the major proteins already described for neuron-derived vesicles. In addition, we have analyzed the content for various amino acids of these vesicles by means of chiral capillary electro- phoresis coupled to laser-induced fluorescence detection and liquid chromatography coupled to mass spectrometry. Post- embedding immunogold labelling of the rat neocortex and hippocampus further revealed the expression of D-serine and glutamate in astrocyte processes contacting excitatory sy- napses. Our results provide significant support for the existence of secretory glial vesicles storing chemical substances like D- serine and glutamate and thus point to the co-release of amino acids by exocytosis in astrocytes.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth and induces encystment in Acanthamoeba castellanii.

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a toxic antimetabolite produced by the opportunistic pathogen Pseudomonas aeruginosa. To evaluate its importance as a potential virulence factor, we tested the host response towards AMB using an Acanthamoeba castellanii cell model. We found that AMB (at concentrations ≥ 0.5 mM) caused amoebal encystment in salt buffer, while inhibiting amoebal growth in rich medium in a dose-dependent manner. However, no difference in amoebal plaque formation was observed on bacterial lawns of wild type and AMB-negative P. aeruginosa strains. We thereby conclude that AMB may eventually act as a virulence factor, but only at relatively high concentrations.

Tumor-reactive, SSX-2-specific CD8+ T cells are selectively expanded during immune responses to antigen-expressing tumors in melanoma patients.

The SSX-2 gene encodes a tumor-specific antigen expressed in neoplasms of various histological types. By analyzing a tumor-infiltrated lymph node of a melanoma patient bearing an SSX-2-expressing tumor, we have recently identified the first SSX-2-derived CD8(+) T-cell epitope, that corresponds to peptide SSX-2(41-49), and is recognized by specific CTL in an HLA-A2 restricted fashion. Here, we have used fluorescent HLA-A2/SSX-2(41-49) peptide multimeric complexes to analyze the response to SSX-2(41-49) in melanoma patients and healthy donors. Multimer(+) CD8(+) T cells were readily detected in the majority of patients bearing SSX-2-expressing tumors and, at lower proportions, in patients with nonexpressing tumors and healthy donors. Importantly, isolated A2/SSX-2(41-49) multimer(+) CD8(+) T cells exhibited a large functional heterogeneity in terms of antigen recognition and tumor reactivity. SSX-2-specific CTLs isolated from tumor-infiltrated lymph node of antigen-expressing patients as well as from the corresponding peripheral blood mononuclear cells exhibited high functional avidity of antigen recognition and efficiently recognized antigen-expressing tumors. In contrast, SSX-2-specific CTLs isolated from patients with undetectable responses in the tumor-infiltrated lymph node, as well as from healthy donors, recognized the antigen with decreased functional avidity and were not tumor reactive. Together, these data indicate that CD8(+) T-cell responses to SSX-2(41-49) frequently occur in SSX-2-expressing melanoma patients and suggest that SSX-2(41-49)-specific CTLs of high avidity and tumor reactivity are selectively expanded during immune responses to SSX-2-expressing tumors in vivo.

An anti-CD19 antibody coupled to a tetanus toxin peptide induces efficient Fas ligand (FasL)-mediated cytotoxicity of a transformed human B cell line by specific CD4+ T cells.

Treatment of B cell lymphoma patients with MoAbs specific for the common B cell marker (CD20) has shown a good overall response rate, but the number of complete remissions is still very low. The use of MoAbs coupled to radioisotopes can improve the results, but induces undesirable myelodepression. As an alternative, we proposed to combine the specificity of MoAbs with the immunogenicity of T cell epitopes. We have previously shown that an anti-Ig lambda MoAb coupled to an MHC class II-restricted universal T cell epitope peptide P2 derived from tetanus toxin induces efficient lysis of a human B cell lymphoma by a specific CD4+ T cell line. Here we demonstrate that the antigen presentation properties of the MoAb peptide conjugate are maintained using a MoAb directed against a common B cell marker, CD19, which is known to be co-internalized with the B cell immunoglobulin receptor. In addition, we provide evidence that B cell lysis is mediated by the Fas apoptosis pathway, since Fas (CD95), but not tumour necrosis factor receptor (TNFr) or TNF-related receptors, is expressed by the target B cells, and FasL, but not perforin, is expressed by the effector T cells. These results show that B cell lymphomas can be 'foreignized' by MoAb-peptide P2 conjugates directed against the common B cell marker CD19 and eliminated by peptide P2-specific CD4+ T cells, via the ubiquitous Fas receptor. This approach, which bridges the specificity of passive antibody therapy with an active T cell immune response, may be complementary to and more efficient than the present therapy results with unconjugated chimeric anti-CD20 MoAbs.

Regulation of the expression of Fit insect toxin locus genes in the root-associated biocontrol pseudomonad CHA0

The root-colonizing Pseudomonas fluorescens strain CHA0 is a biocontrol agent of soil-borne plant diseases caused by fungal and oomycete pathogens. Remarkably, this plant-beneficial pseudomonad is also endowed with potent insecticidal activity that depends on the production of a large protein toxin termed Fit (for P. fluorescens insecticidal toxin). In our present work, the genomic locus encoding the P. fluorescens insect toxin is subjected to a detailed molecular analysis. The Fit toxin gene fitD is flanked upstream by the fitABC genes and downstream by the fitE gene that encode the ABC transporter, membrane fusion, and outer membrane efflux components of a type I protein secretion system predicted to function in toxin export. The fitF, fitG, and fitH genes located downstream of fitE code for regulatory proteins having domain structures typical of signal transduction histidine kinases, LysR-type transcriptional regulators, and response regulators, respectively. The role of these insect toxin locus-associated control elements is being investigated with mutants defective for the regulatory genes and with GFP-based reporter fusions to putative promoter regions upstream of the transporter genes fitA and fitE, the toxin gene fitD, and the regulatory genes fitF and fitH. Our preliminary findings suggest that the three regulators interact with known global regulators of biocontrol factor expression to control Fit toxin expression and secretion.

Characterization of functional role of α-ketoglutarate receptor in the kidney : role of the intrinsic circadian timing system in renin expressing cells

Le rein joue un rôle essential dans le maintien de l'homéostasie des fluides extracellulaires (FEC) et la pression artérielle. L'objectif de notre groupe est d'identifier de nouveaux mécanismes impliqués dans le contrôle de l'homéostasie des FEC et de la pression artérielle par le rein. Projet 1) Caractérisation du rôle fonctionnel du récepteur à l'a-cétogluatarate Oxgrl dans le rein Oxgrl est le récepteur spécifique de l'a-cétogluatarate, une moléule intermédiaire du cycle de l'acide citrique, filtrée par le rein et réabsorbée ou secrétée au niveau des tubules proximaux. Le rôle fonctionnel de ces deux récepteurs reste inconnu. Nos résultats montrent qu'Oxgrl est localisé au niveau des cellules intercalaires du tube collecteur (CCD). Des souris (Oxgrr/_) montrent une diminution du pH urinaire ,une augmentation de la concentration de l'acide urinaire titrable et une augmentation des niveaux d'a-cétoglutarate. Le traitement au Na-bicarbonate provoque une augmentation plus prononcée de l'alcalose métabolique chez les souris Oxgrl"7"' accompagnée d'une augmentation de la concentration de bicarbonate et une diminution du niveau de chlore plasmatique. En parallèle, des études de microperfusion ont montré que a-cétoglutarate stimule la réabsorption éléctroneutre de NaCl dans le CCD des souris de type sauvage mais pas des souris Oxgrl"7". En résumé, ces résultats montrent que l'a-cétoglutarate joue un rôle de molécule messagère du tubule proximal jusqu'au tube collecteur au niveau du rein et qu'Oxgrl pourrait être impliqué dans la régulation de l'échange Cl/bicarbonate et la réabsorption du NaCl dans les cellules intercalées. Projet 2) Rôle du système circadien dans les cellules productrices de rénine. Le système chronologique circadien est un mécanisme moléculaire ubiquitaire qui permet à l'organisme de coordonner ses fonctions principales en fonction du temps géophysique. Comme l'activité de la rénine plasmatique montre une rythmicité circadienne nette chez l'homme et la souris ; dans ce projet, nous avons abordé la question à savoir dans quelle mesure le système circadien est impliqué dans cette variabilité circadienne. Pour cela, le gène Bmall, élément principal de l'horloge moléculaire, a été perturbé dans les cellules granulaires productrices de rénine par le système Cre-LoxP. Nos résultats montrent que les souris Renld- Cre/Bmalllox/lox (cKO) présentent de faibles taux d'ARNm de Reni, altèrent la dynamique d'expression de la protéine rénine, mais il le niveau de concentration plasmatique de la rénine reste le même. Cependant, les souris cKO montrent une réduction significative de la concentration plasmatique de l'aldostérone. Nos analyses de l'urine récupérée dans des intervalles de temps de 24 et 1 heure montrent une augmentation du volume urinaire, une tendance à une hypercalciurie, ainsi qu'une altération de la dynamique d'excrétion urinaire de sodium chez les souris cKO. Plusieurs gènes impliqués dans la production/sécrétion de la rénine et dans le contrôle de la fonction rénale montrent une altération de l'expression circadienne d'ARNm. Par ailleurs, les souris cKO montrent une baisse significative de la pression artérielle. Nos résultats suggèrent que l'horloge intrinsèque des cellules productrices de la rénine joue un rôle important dans le control des FEC et l'homéostasie de la pression artérielle via régulation de la fonction rénale.

The effect of vaccines based on ovalbumin coupled to gas-filled microbubbles for reducing infection by ovalbumin-expressing Listeria monocytogenes.

Gas-filled microbubbles (MB) are a very promising alternative to the currently evaluated lipid- or polymer-based particulate Ag delivery systems. We recently demonstrated the ability of MB to deliver associated Ag to DC, to activate them and thereby induce both humoral and cellular immune responses. We now extended the characterization of MB as antigen-delivery system by appraising the efficiency of MB-associated ovalbumin (OVA-MB) at protecting mice against pathogen infection. Ultrasound-mediated imaging demonstrated that the administration of OVA via MB generates a depot at the injection site that lasts for several hours. We found that OVA-MB injected subcutaneously is far more effective at inducing specific Ab and T cell immunity than immunization with free OVA. Moreover, a covalent link between MB and OVA causes a stronger bias towards a Th1-type of immune response than adsorption of the Ag or its covalent link to liposomes of the same lipid composition. Finally, vaccination of mice with OVA-MB partially protects against a systemic infection with OVA-expressing Listeria monocytogenes. The vaccine induces specific effector CD8 T cell responses capable of decreasing more than 100 fold the bacterial load. MB thus represent a potent Ag delivery system for vaccination against intracellular infectious agents.

Study of the mechanisms regulating the proliferative potential of human CD8+T lymphocytes over-expressing telomerase reverse transcriptase (hTERT)

Résumé La plupart des cellules issues du sang ont une durée de vie limitée. Dans les cellules somatiques humaines, y incluant les lymphocytes T, la taille des télomères diminue progressivement à chaque division cellulaire, pouvant aboutir à des instabilités chromosomiques. L'expression ectopique du gène de la transcriptase réverse de la télomérase (hTERT) dans les cellules restaure l'activité de la télomérase, et permet un rallongement de leur vie réplicative. Malgré l'absence de signes caractéristiques de transformation, nous ne savons pas encore si les cellules somatiques qui surexpriment hTERT sont physiologiquement indiscernables des cellules normales. Certaines études récentes proposent que la télomérase joue plusieurs rôles additionnels dans d'autres phénomènes biologiques tels que la réparation de l'ADN, la survie et la croissance des cellules. Dans notre étude, nous avons utilisé des clones issus de lymphocytes T cytotoxiques surexprimant la télomérase afin d'étudier les mécanismes moléculaires qui règlent leur prolifération et leur sénescence. Nous avons montré que les «jeunes » cellules T exprimant ou non hTERT révèlent des taux de croissance identiques suite à des réponses de stimulation induites par des mitogènes. De plus, aucun changement global dans leur expression des gènes n'a pu être mis en évidence. Curieusement, nous avons observé des réponses réduites dans la prolifération des cellules transduites avec la télomérase qui présentaient une élongation des télomères et une durée de vie prolongée. Ces cellules, malgré le maintien d'un niveau élevé de l'expression de gènes impliqués dans la progression du cycle cellulaire, ont également montré une expression accrue de plusieurs gènes trouvés en commun avec nos lymphocytes T vieillissants n'exprimant pas de télomérase. En particulier, les cellules ayant une durée de vie prolongée grâce à l'expression de la télomérase accumulaient également certains inhibiteurs du cycle cellulaire tels que p16Ink4a et p21Cip1, associés à l'arrêt de la croissance cellulaire. En résumé, nos résultats indiquent la présence fonctionnelle de mécanismes alternatifs pouvant contrôler la croissance réplicative de ces cellules; ils sont donc encourageants dans l'optique d'une utilisation à moindre risque de lymphocytes T «immortalisés » à des fins thérapeutiques pour traiter les tumeurs malignes ou les infections. Summary Most mature blood cells have a finite life span. In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division eventually leading to chromosomal instability. Ectopic expression of the human telomerase reverse transcriptase (hTERT) gene in cells restores telomerase activity and results in the extension of their replicative life span. Despite lack of transformation characteristics, it is yet unknown whether somatic cells that over-express telomerase are biologically and physiologically indistinguishable from normal cells. Recent data suggest that telomerase might mediate additional functions in DNA repair, cell survival and cell growth. Using CD8+ T lymphocyte clones over-expressing telomerase we investigated the molecular mechanisms that regulate T cell proliferation and senescence. Here we show that early-passage T cell clones transduced or not with hTERT displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in hTERT-transduced cells with elongated telomeres and extended life span. These cells, despite maintaining high expression level of genes involved in cell cycle division and progression, also showed increased expression of several genes associated with normal aging T lymphocytes. In particular, late passage T cells over-expressing telomerase accumulated the cyclin-dependent inhibitors p16INK4a and p21CIP1 that have largely been associated with in vitro growth arrest. Whether tumor-reactive CD8+ T cells that ectopically express telomerase could now be used for adoptive transfer therapy in cancer patients remains unclear at this point. Nevertheless, our results regarding the safe and effective use of hTERT-transduced lymphocytes are encouraging, since they indicate that alternative growth arrest mechanisms such as p 16 and p21 are still functional in these cells and regulate to some extend their growth potential.

Indoleamine 2,3-dioxygenase-expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells.

A comprehensive understanding of the complex, autologous cellular interactions and regulatory mechanisms that occur during normal dendritic cell (DC)-stimulated immune responses is critical to optimizing DC-based immunotherapy. We have found that mature, immunogenic human monocyte-derived DCs (moDCs) up-regulate the immune-inhibitory enzyme, indoleamine 2,3-dioxygenase (IDO). Under stringent autologous culture conditions without exogenous cytokines, mature moDCs expand regulatory T cells (Tregs) by an IDO-dependent mechanism. The priming of resting T cells with autologous, IDO-expressing, mature moDCs results in up to 10-fold expansion of CD4(+)CD25(bright)Foxp3(+)CD127(neg) Tregs. Treg expansion requires moDC contact, CD80/CD86 ligation, and endogenous interleukin-2. Cytofluorographically sorted CD4(+) CD25(bright)Foxp3(+) Tregs inhibit as much as 80% to 90% of DC-stimulated autologous and allogeneic T-cell proliferation, in a dose-dependent manner at Treg:T-cell ratios of 1:1, 1:5, and as low as 1:25. CD4(+)CD25(bright)Foxp3(+) Tregs also suppress the generation of cytotoxic T lymphocytes specific for the Wilms tumor antigen 1, resulting in more than an 80% decrease in specific target cell lysis. Suppression by Tregs is both contact-dependent and transforming growth factor-beta-mediated. Although mature moDCs can generate Tregs by this IDO-dependent mechanism to limit otherwise unrestrained immune responses, inhibition of this counter-regulatory pathway should also prove useful in sustaining responses stimulated by DC-based immunotherapy.

Larval development of Spodoptera eridania (Cramer) fed on leaves of Bt maize expressing Cry1F and Cry1F + Cry1A.105 + Cry2Ab2 proteins and its non-Bt isoline

This study aimed to evaluate, in controlled laboratory conditions (temperature of 25±2 °C, relative humidity of 60±10%, and 14/10 h L/D photoperiod), the larval development of Spodoptera eridania (Cramer, 1784) (Lepidoptera, Noctuidae) fed with leaves of Bt maize expressing Cry1F and Cry1F + Cry1A.105 + Cry2Ab2 insecticide proteins and its non-Btisoline. Maize leaves triggered 100% of mortality on S. eridania larvae independently of being Bt or non-Bt plants. However, it was observed that in overall Bt maize (expressing a single or pyramided protein) slightly affects the larval development of S. eridania, even under reduced leaf consumption. Therefore, these results showed that Cry1F and Cry1F + Cry1A.105 + Cry2Ab2 can affect the larval development of S. eridania, although it is not a target pest of this plant; however, more research is needed to better understand this evidence. Finally, this study confirms that non-Bt maize leaves are unsuitable food source to S. eridania larvae, suggesting that they are not a potential pest in maize fields.

Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells.

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8(+) T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8(+) cells were observed in the periphery. Upon specific activation, transgenic CD8(+) T cells efficiently lysed syngeneic MHC class I(+) targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

Contribution of PaTrin-2 in Radiotherapy with Temozolomide for the Treatment of Glioblastoma Expressing MGMT

Purpose/Objective(s): Current standard treatment of glioblastoma is radiotherapy (RT) concomitant with temozolomide (TMZ), an alkylating agent. O6-methylguanine-DNA methyltransferase (MGMT) expression is a major mechanism of resistance to Proceedings of the alkylating agent chemotherapy, and MGMT gene promoter methylation (present in 30-45 % of tumors) has been shown to be predictive for tumor response to TMZ therapy. MGMT, an exhaustible repair protein can be depleted by specific inhibitors such as O6- benzylguanine or the non-toxic O6-(4-bromothenyl)guanine (PaTrin-2). Here we have studied the efficacy of the combination of TMZ, RT, and PaTrin-2 to improve the treatment outcome in glioblastoma expressing MGMT. Materials/Methods: 3 glioblastoma lines were chosen: LN18 and T98G expressing MGMT and U251 lacking MGMT expression. A shRNA approach was used to selectively and permanently knockdown level of MGMT in LN18 line. Cells were treated with 10 mM PaTrin-2. After 2 h, various concentrations of TMZ were added, cells were incubated for 24 h, and clonogenic assays were performed. After the same PaTrin-2 pretreatment and 100 mM TMZ exposure, cells were plated 4 h before irradiation with increasing RT doses of up to 6 Gy. Clonogenic survival was assessed after 14 days. Results: Western blot analysis confirmed that reduction of MGMT expression was achieved in LN18A1 expressing MGMT-targeting shRNA. The shRNA non-targeting control sequence did not influenceMGMTprotein level (LN18NT). PaTrin-2 showed no toxicity at 10 mMon the 5 cell lines. TMZ induced up to 70 and 97%of cell death on LN18A1 and U251, respectively, but was not toxic up to 50 mMfor T98G, LN18, and LN18NT. Up to 53%increased TMZ toxicity was observed on the 5 cell lines when treated with the 2 drugs. Irradiation of the 5 lines treated or not with PaTrin-2 showed no survival difference at any irradiation dose. When LN18A1 and U251 cells were irradiated post TMZ treatment, an up to 2.5 and 139.4 fold increase in toxicity, respectively, was observed compared to un-pretreated controls. By contrast, TMZ pretreatment did not increase irradiation toxicity on T98G, LN18, and LN18NT. When cells were incubated with PaTrin-2 and TMZ before the irradiation, up to 3.7, 3.9, 5.8, 6.6 and 348.5 fold increase in toxicity was observed compared to controls on LN18, LN18NT, LN18A1, T98G and U251, respectively. Conclusions: We present here results of TMZ and PaTrin-2 combination ± RT on glioblastoma lines. U251 and LN18A1 cells were much more sensitive to TMZ than LN18, LN18NT, and T98G. PaTrin-2 enhanced the toxicity of TMZ on the MGMT expressing glioblastoma lines. RT further increased TMZ and PaTrin-2 efficacy. These results are encouraging for the treatment of patients with glioblastoma expressing MGMT who have the worst prognosis and respond poorly to RT combined with TMZ.

Occurrence and molecular diversity of the Fit insect toxin locus in plant-beneficial pseudomonads

The application of plant-beneficial pseudomonads provides a promising alternative to chemical pest management in agriculture. The fact that Pseudomonas fluorescens CHA0 and Pf-5, both well-known biocontrol agents of fungal root diseases, exhibit also potent insecticidal activity is of particular interest, as these plant-beneficial bacteria naturally colonize the rhizosphere of important crop plants. Insecticidal activity in these strains depends on a novel locus encoding the production of a protein toxin termed Fit (for P. fluorescens insecticidal toxin). To gain a better understanding of the ecological relevance of the Pseudomonas anti-insect activity, we have begun to investigate the occurrence and molecular diversity of the Fit toxin genes among root-associated pseudomonads. To this end, we have screened a large world-wide collection of fluorescent Pseudomonas sp. isolated from the roots of different plant species using molecular fingerprinting techniques. The strains are already well characterized for exoproduct patterns and disease-suppressive ability and are currently being tested for insecticidal activity in a greater wax moth larvae assay system.