[Identification of proteins associated with transcription factors HOXA9 and E2A-PBX1 by tandem affinity purification].

Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 might indicate a novel function for HOX proteins independent of their transcriptional activity.

Carabid assemblages (Coleoptera: Carabidae) in a rotation of three different crops in southern Alberta, Canada: a comparison of sustainable and conventional farming.

Carabids were sampled in 2000 (pretreatment year) and 2003-2005 in experimental plots in southern Alberta, Canada, after a rotation of beans, wheat, and potato under sustainable and conventional farming practices. Each phase of the rotation was present in every year. Crop type had a stronger effect than sustainable treatment on carabid-expected species richness, diversity, and species composition. However, carabid activity density was consistently higher in plots under sustainable treatments than those maintained conventionally. Potato plots, which were sprayed with insecticide for pest control, showed a significantly lower carabid activity density than the other crops. These results support other studies showing the beneficial effect of sustainable farming on activity density of carabid beetles.

Response of ground beetle (Coleoptera: Carabidae) field populations to four years of Lepidoptera-specific Bt corn production.

Pitfall traps were used to monitor populations of ground beetles (Coleoptera: Carabidae) in plots of corn grown in continuous cultivation during a 4-yr period (2000-2003). Treatments included transgenic corn expressing a Bt Cry protein with efficacy specific against Lepidoptera (Bt), conventional corn grown with insecticide application (I), and the same conventional cultivar grown without insecticide application (NI). Mixed-model analyses of variance were performed on pitfall captures of beetles combined across weeks to give seasonal sums. Effects of corn treatment were not detected (P > 0.05) on total beetle abundance or species richness in any year. Effects of corn treatment on individual taxa were detected (P < 0.05) for 3 of the 39 species-by-year combinations examined. Effects of near significance (P < 0.08) were detected for an additional two species. In 2001, captures of Amara farcta Leconte and Harpalus amputatus Say were lower in Bt plots than in I or NI plots. In 2003, captures of Amara apricaria (Paykull) and Amara carinata (Leconte) were higher in Bt plots than in I or NI plots. Also in 2003, captures of Poecilus scitulus Leconte were higher in I plots than in Bt or NI plots. These patterns were not repeated among years. Results of this study indicate that cultivation of Lepidoptera-specific Bt corn in southern Alberta does not appreciably affect ground beetle populations.

Identification of Streptomyces coelicolor proteins that are differentially expressed in the presence of plant material.

Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.

Preferential perinuclear localization of poly(ADP-ribose) glycohydrolase.

The transient nature of poly(ADP-ribosyl)ation, a posttranslational modification of nuclear proteins, is achieved by the enzyme poly(ADP-ribose) glycohydrolase (PARG) which hydrolyzes the poly(ADP-ribose) polymer into free ADP-ribose residues. To investigate the molecular size and localization of PARG, we developed a specific polyclonal antibody directed against the bovine PARG carboxy-terminal region. We found that PARG purified from bovine thymus was recognized as a 59-kDa protein, while Western blot analysis of total cell extracts revealed the presence of a unique 110-kDa protein. This 110-kDa PARG was mostly found in postnuclear extracts, whereas it was barely detectable in the nuclear fractions of COS7 cells. Further analysis by immunofluorescence revealed a cytoplasmic perinuclear distribution of PARG in COS7 cells overexpressing the bovine PARG cDNA. These results provide direct evidence that PARG is primarily a cytoplasmic enzyme and suggest that a very low amount of intranuclear PARG is required for poly(ADP-ribose) turnover.

Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo.

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.

Poly(ADP-ribose) glycohydrolase is present and active in mammalian cells as a 110-kDa protein.

Poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme responsible for the catabolism of poly(ADP-ribose), a reversible covalent-modifier of chromosomal proteins. Purification of PARG from many tissues revealed heterogeneity in activity and structure of this enzyme. To investigate PARG structure and localization, we developed a highly sensitive one-dimensional zymogram allowing us to analyze PARG activity in crude extracts of Cos-7, Jurkat, HL-60, and Molt-3 cells. In all extracts, a single PARG activity band corresponding to a protein of about 110 kDa was detected. This 110-kDa PARG activity was found mainly in cytoplasmic rather than in nuclear extracts of Cos-7 cells.

Lipid concentration profile across the wall of pseudoatherosclerotic synthetic arterial prostheses using FTIR microspectroscopy.

FTIR microscopy is a versatile technique successfully used to probe the subcellular chemical composition of atherosclerostic arterial walls. To design new vascular substitutes that resist lipid uptake (the major cause of the phenomenon referred to as atherosclerosis-like), identifying and understanding lipid distribution within the pseudoatherosclerosed arterial prostheses is of prime importance. Until now, the amount of lipids present within arterial prostheses that had been explanted from either animals (during in vivo trials) or humans (after the failure of vascular grafts) or had been submitted to in vitro investigations could only be measured through the use of histological techniques or radioactive labeling methods. We present here a novel method to quantitatively measure the lipid concentration profile within the wall of arterial prostheses by means of Fourier transform infrared microspectroscopy. Essentially, prostheses are fixed in a 1% osmium tetraoxide aqueous solution under vacuum and radially cut with a 5-micron thickness with a microtome. The sections are then placed onto BaF2 windows and observed with a microscope attached to a FTIR spectrometer with a 30 microns x 50 microns sampling area. The lipid concentration profile is obtained by scanning the prosthesis wall from the inner to the outer surface and reporting the corresponding integrated absorbance between 2700 and 3100 cm(-1) against a calibration curve. The application of this technique constitutes the first quantitative measurement of the concentration of biological molecules within the wall of artificial arterial substitute.

Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a highly conserved nuclear enzyme present in higher eukaryotes. PARP is activated following DNA damage, is implicated in DNA repair, and its proteolysis has been shown to be an early marker of programmed cell death or apoptosis. In order to better understand the role of PARP in apoptosis and DNA repair and also to study PARP automodification, we have developed anti-peptide sera directed against four peptides from the conserved automodification domain of PARP. Four peptides were synthesized according to the four branched Multiple Antigenic Peptide (MAP) system and injected into rabbits. Immune sera were titrated by ELISA and analysed in Western blotting experiments on cell lines. The sera were also analysed for their capacity to inhibit PARP activity in an in vitro assay. Of the eight sera developed (two for each peptide), a serum directed against a peptide localized at the C-terminal part of the automodification domain of PARP (#422) appeared to be the best antibody to detect PARP from different species. All antipeptide antibodies were efficient in detecting the apoptotic fragment of PARP during programmed cell death in HL-60 apoptotic cells. None of the serum alone was able to completely inhibit PARP activity but combinations of the sera could significantly reduce automodification of PARP consistent with the localization of half of the automodification sites on bovine PARP. Sera were also used to map proteolysed purified PARP and to immunoprecipitate purified bovine PARP.

Characterization of antibodies specific for the caspase cleavage site on poly(ADP-ribose) polymerase: specific detection of apoptotic fragments and mapping of the necrotic fragments of poly(ADP-ribose) polymerase.

Intracellular cysteine proteases are important mediators of apoptosis. Indeed, some nuclear proteins and enzymes are cleaved during apoptosis, in particular poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand interruptions and is involved in DNA repair. PARP is cleaved into two fragments of 29 and 85 kDa (apparent molecular mass) in human promyelomonocytic leukemia cells, HL-60, treated with etoposide to induce apoptosis. These cells possess protease activities, caspases, that share many features with the ICE/CED-3 family. The cleavage occurs between Asp-214 and Gly-215, a site that is conserved in human, bovine, and chicken PARP. This cleavage has been shown to be an early marker of apoptosis. To monitor apoptosis, to understand the role of PARP cleavage by caspases, and to study the role of the two fragments in DNA repair, members of our laboratory have developed two polyclonal antipeptide antibodies directed against the two human PARP sequences: [196-214] for LP96-22 and [215-228] for LP96-24. Moreover, these antibodies will be useful to map the necrotic cleavage of PARP, which generates fragments different from those obtained during apoptosis, and thus to discriminate between apoptotic and necrotic cell death.

Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres.

The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly [2], but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A [3], a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CSE4 [6]. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes [7], our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA [8]. However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.

Protection of islets of Langerhans from antibodies by microencapsulation with alginate-poly-L-lysine membranes.

Microencapsulation of islets has been proposed to prevent their immune destruction following transplantation. An indirect immunofluorescence technique has been developed and used to study the permeability of the alginate-poly-L-lysine microcapsules to antibodies. Wistar rat islets were incubated with the R2D6 monoclonal mouse IgM antibody against rat islets, microencapsulated, and incubated with fluorescein-labeled goat IgG antibodies against mouse IgG and IgM. For the negative controls, the first antibody was omitted or both antibodies were omitted. The positive controls included islets incubated with both antibodies before they were encapsulated. Our study demonstrated that the alginate-poly-L-lysine membranes are not permeable to IgG when poly-L-lysine of molecular weights ranging from 21,000 to 390,000 are used. This simple immunofluorescence technique demonstrated the nonpermeability of the microcapsules to IgG, and could be useful for the initial evaluation of new types of membranes.

Expression of an inducible enzyme II fructose and activation of a cryptic enzyme II glucose in glucose-grown cells of spontaneous mutants of Streptococcus salivarius lacking the low-molecular-mass form of IIIman, a component of the phosphoenolpyruvate:mannose phosphotransferase system.

We have reported previously that the phosphoenolpyruvate:mannose phosphotransferase system (mannose PTS) of Streptococcus salivarius, consisting of an Enzyme II mannose (EIIman) and two forms of Enzyme III mannose (IIIman) with Mr values of 38,900 and 35,200, respectively, concomitantly transports and phosphorylates mannose, as well as glucose and fructose. In this paper, we report the presence, in S. salivarius, of alternative specific fructose and glucose PTSs encoded by inducible and cryptic genes, respectively. Protein phosphorylation experiments conducted with [32P]phosphoenolpyruvate have allowed us to identify by SDS-PAGE and autoradiography the EII fructose (EIIfru) (Mr 57,500) and the EII glucose (EIIglc) (Mr 58,700). No proteins corresponding to IIIfru or IIIglc could be detected. EIIfru phosphorylated fructose on the C-1 position rather than, as with the constitutive mannose PTS, on the C-6 position. Growth on fructose resulted in the induction of EIIfru as well as an increase of 1-phosphofructokinase activity. Nevertheless, the genes encoding these proteins were independently regulated. Studies carried out with spontaneous mutants lacking the low-molecular-mass form of IIIman (mutants A37, G29 and B31) showed that EIIfru was expressed in glucose-grown cells of strains G29 and B31, but not in strain A37, whereas the cryptic gene encoding EIIglc was activated in all three mutant strains. The results obtained with the mutants suggest that the three spontaneous mutants were not all mutated on the gene encoding IIIman although all of them lacked IIIman.

Effect of humidity on the deswelling function of the human cornea.

The present study determined the effect of the full range of humidities on the deswelling function of the human cornea. The closed-eye deswelling function and the open-eye deswelling responses for five different levels of humidity (0%, 25%, 60%, 85%, and 100%) were assessed for 8 normal, young-adult subjects. Open-eye corneal deswelling for the 8 subjects was unaffected by relative humidities from 0 to 100%. Therefore, osmotically-driven corneal thinning effect of tear evaporation does not significantly contribute to the deswelling function of the human cornea. We conclude, contrary to recent reports, that the endothelial pump is the primary mechanism that maintains normal corneal thickness and provides recovery from stromal edema.

Control of sugar utilization in oral streptococci. Properties of phenotypically distinct 2-deoxyglucose-resistant mutants of Streptococcus salivarius.

The physiological and biochemical characterization of Streptococcus salivarius mutants isolated by positive selection for resistance to 0.5 mM 2-deoxyglucose in the presence of lactose are reported. We found 2 classes of mutants following a series of experiments that included: growth rate determinations, uptake studies, measurement of phosphotransferase system (PTS) activities and detection of the IIIman proteins by Western blotting and analysis of [32P]PEP-phosphorylated proteins. Class 1 mutants did not possess the low-molecular-weight form of IIIman. They did not grow on mannose and were unable to transport 2-deoxyglucose. On the other hand, class 2 mutants possessed the 2 forms of IIIman, grew readily on mannose and transported 2-deoxyglucose, albeit at a lower rate than the parental strain. Both classes of mutants exhibited abnormal growth in media containing mixtures of sugars. Moreover, derepression of genes coding for catabolic enzymes was observed in all the mutant strains. Our data suggested that the role of the mannose PTS in the control of sugar utilization in S. salivarius is complex and may involve the participation of several components.

A IIIman protein is involved in the transport of glucose, mannose and fructose by oral streptococci.

We show in this article that the transport of glucose, mannose and fructose by the phosphoenolpyruvate: mannose phosphotransferase system of oral streptococci requires the participation of a protein component that we have called IIIman. This protein was purified from Streptococcus salivarius by chromatography on DEAE-cellulose, DEAE-TSK, hydroxyapatite, and Dyematrex Green A. The purified protein migrated as a 38,900 molecular weight protein on a sodium dodecyl sulfate polyacrylamide gel. However, electrophoretic analysis of phosphoproteins and Western blot experiments indicated the presence in membrane-free cellular extracts of S. salivarius of 2 different forms of IIIman having molecular weights of 38,900 and 35,200. The presence of the high-molecular-weight form of IIIman was observed by immunodiffusion, Western blot and phosphorylation by [32]PEP in S. salivarius, Streptococcus mutans, Streptococcus sobrinus, and Streptococcus lactis but not in Streptococcus faecium, Staphylococcus aureus, Bacillus subtilis and Lactobacillus casei. Antibodies directed against the IIIman of S. salivarius did not react with the IIIman of Escherichia coli.