Nice to meet you: genetic, epigenetic and metabolic controls of plant perception of beneficial associative and endophytic diazotrophic bacteria in non-leguminous plants.

A wide range of rhizosphere diazotrophic bacteria are able to establish beneficial associations with plants, being able to associate to root surfaces or even endophytically colonize plant tissues. In common, both associative and endophytic types of colonization can result in beneficial outcomes to the plant leading to plant growth promotion, as well as increase in tolerance against biotic and abiotic stresses. An intriguing question in such associations is how plant cell surface perceives signals from other living organisms, thus sorting pathogens from beneficial ones, to transduce this information and activate proper responses that will finally culminate in plant adaptations to optimize their growth rates. This review focuses on the recent advances in the understanding of genetic and epigenetic controls of plant-bacteria signaling and recognition during beneficial associations with associative and endophytic diazotrophic bacteria. Finally, we propose that "soil-rhizosphere-rhizoplane-endophytes-plant" could be considered as a single coordinated unit with dynamic components that integrate the plant with the environment to generate adaptive responses in plants to improve growth. The homeostasis of the whole system should recruit different levels of regulation, and recognition between the parties in a given environment might be one of the crucial factors coordinating these adaptive plant responses.

Signaling and drug sensitivity.

Even though alterations in receptor and nonreceptor kinases are involved in the development of human cancer, many cancer cell lines still retain their responsiveness to growth factors. We have investigated the hypothesis that cellular signaling events regulate the sensitivity of cancer cells to chemotherapeutic agents. In 2008 human ovarian carcinoma cells, activation of a number of different transduction pathways resulted in a 2 to 4-fold increase in the sensitivity to cisplatin. These signaling events include pathways activated by the epidermal growth factor (EGF) receptor, tumor necrosis factor alpha (TNF alpha) receptor, bombesin receptor, protein kinase A (PKA), and protein kinase C (PKC). Enhanced sensitivity to chemotherapeutic agents is presumed to be mediated by phosphorylation of critical target protein(s). beta-tubulin has been identified as one such target for the protein kinase signaling cascade. For other signal transduction pathways the key substrates that regulate drug sensitivity have not yet been identified. Recent work has shown that DNA damaging agents activate signaling cascades one of which involves the Src, Ras, and Raf proteins as intermediates and results in induction of a number of genes, including c-fos, c-jun, and the growth arrest and DNA damage-inducible (gadd) genes. This signaling cascade has been shown to involve activation of protein kinase C and to have a protective function. With the growing understanding of how signaling events relate to damage response and drug sensitivity, new and potentially useful strategies for modulating drug sensitivity are evolving.

Cisplatin sensitivity correlates with its ability to cause cell cycle arrest via a wee1 kinase-dependent pathway in Schizosaccharomyces pombe.

Mutants of Schizosaccharomyces pombe were used to define genes involved in the cell cycle arrest produced by cisplatin (DDP), an agent that causes both DNA damage and inhibition of DNA synthesis. Previous work has demonstrated that strains with defective or absent wee1+ function fail to arrest in G2 when DNA is damaged, but do arrest when DNA synthesis is inhibited (Rowley et al., 1992a, Nature, 356:353-355). Strains defective in wee1+ function, or in the ability of the wee1+ kinase to regulate cdc2, failed to arrest following DDP exposure, as did a rad1-1 mutant. All strains failing to arrest in G2 were hypersensitive to DDP. Thus, DNA damage rather than inhibition of DNA synthesis is causative of DDP-induced cell cycle arrest. In addition, this work shows that the wee1+ and rad1+ gene products are required for successful DDP-induced arrest, and suggests that the ability of S. pombe to arrest is a major determinant of sensitivity to DDP.

In vitro modulation of cisplatin accumulation in human ovarian carcinoma cells by pharmacologic alteration of microtubules.

We have previously shown that forskolin and 3-isobutyl-1-methylxanthine (IBMX) increased accumulation of cisplatin (DDP) in DDP-sensitive 2008 human ovarian carcinoma cells in proportion to their ability to increase cAMP. Since the major function of cAMP is to activate protein kinase A, it was conjectured that the stimulation of DDP accumulation was mediated by a protein kinase A substrate. We now show that exposure of 2008 cells to forskolin resulted in phosphorylation of a prominent 52-kD membrane protein. Microsequencing of the band demonstrated it to be human beta-tubulin. Similarly, pretreatment of 2008 cells with the microtubule stabilizing drug taxol increased platinum accumulation in a dose-dependent manner. In 11-fold DDP-resistant 2008/C13*5.25 cells, decreased DDP accumulation was associated with enhanced spontaneous formation of microtubule bundles and decreased expression of beta-tubulin and the tubulin-associated p53 antioncogene relative to 2008 cells. 2008/C13*5.25 cells had altered sensitivity to tubulin-binding drugs, being hypersensitive to taxol and cross-resistant to colchicine. We conclude that pharmacologic alterations of tubulin enhance accumulation of DDP, and that the DDP-resistant phenotype in 2008/C13*5.25 cells is associated with tubulin abnormalities.

Evidence for binding of extrachromosomal DNA sequences to nuclear matrix proteins in multidrug-resistant KB-V1 cells.

Multidrug-resistant KB-V1 cells carry amplified mdrl gene sequences located in an extrachromosomal compartment (on episomes). Since episomes do not contain centromeric or telomeric sequences it is unclear whether they are able to bind to nuclear matrix proteins that may regulate episomal gene expression. Using high salt treatments followed by in situ hybridization and dot blot analyses we found evidence for direct binding of episomal DNA to nuclear matrix proteins. This binding could only be reversed after incubation with trypsin or proteinase K as determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Our findings are consistent with the concept that circular extrachromosomal DNA may not only reintegrate into nuclear DNA but may also be subject to functional control by regulatory proteins within the nuclear matrix.

Regulation of HSP60 mRNA expression in a human ovarian carcinoma cell line.

The expression of the 60-kDa heat-shock protein (HSP60) varies markedly among patients with ovarian carcinoma, and high-level expression predicts poor survival in such patients treated with cisplatin (DDP)-containing chemotherapy programs. We investigated the expression of HSP60 in human ovarian carcinoma 2008 cells and an 11-fold DDP-resistant subline 2008/C13*5.25. Heating for 2 h at 44 degrees C produced a 2.7 +/- 0.16-fold increase (mean +/- SD) that was maximal at 4 h after the start of heat exposure. Exposure to an IC50 concentration of DDP for 1 h induced a 1.8 +/- 0.03-fold increase in hsp60 expression. The opposite was true for cadmium and zinc, both of which induced increases in metallothionein IIA but not in the hsp60 message. 2008/C13*5.25 cells constitutively over-expressed hsp60 mRNA by 1.7 +/- 0.16 orders of magnitude and contained a 3.8 +/- 0.45-fold higher level of HSP60 as detected by immunocytochemical staining. 2008/C13*5.25 cells showed 1.2-fold cross-resistance to thermal killing. Expression of hsp60 was markedly reduced in 2008 xenografts as compared with 2008 cells growing in vitro; however, neither serum starvation nor refeeding altered the message level. Exposure to a variety of growth factors and drug treatments known to alter the DDP sensitivity of 2008 cells, including epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, buthionine sulfoximine, ouabain, and forskolin, did not alter hsp60 expression. These results suggest a role for HSP60 in mediating resistance to both DDP and hyperthermia but indicate that the hsp60 mRNA levels are not regulated by the factors listed above.

Modulation of cisplatin sensitivity and growth rate of an ovarian carcinoma cell line by bombesin and tumor necrosis factor-alpha.

A twofold change in the cisplatin (DDP) sensitivity of 2008 human ovarian carcinoma cells is sufficient to reduce tumor response in vivo. The DDP sensitivity of these cells can be enhanced by activation of the epidermal growth factor and protein kinase C signal transduction pathways. We report here that two endogenous growth factors, bombesin and tumor necrosis factor alpha (TNF alpha), enhanced DDP sensitivity by factors of 1.7 +/- 0.1 (SD)-fold and 1.8 +/- 0.1 (SD)-fold, respectively. Both agents also produced sensitization in an 11-fold DDP-resistant 2008 subline. Neither bombesin nor TNF alpha changed the accumulation of DDP, glutathione content, or glutathione-S-transferase activity in 2008 cells. However, a 2-h exposure to both bombesin and TNF alpha was sufficient to increase 2008 cloning efficiency by up to 2.6 +/- 0.1 (SD)-fold and 2.2 +/- 0.1 (SD)-fold, and it increased average colony size by 1.35 +/- 0.1 (SD)-fold and 1.55 +/- 0.1 (SD)-fold, respectively. Bombesin increased intracellular free calcium, and this was blocked by the bombesin receptor-specific antagonist SC196, demonstrating that 2008 cells have functional bombesin receptors. These results indicate that bombesin and TNF alpha can enhance sensitivity to DDP in both DDP sensitive and resistant variants of a human ovarian carcinoma and that both agents serve as growth factors for this tumor.

Expression of the c-Ha-ras oncogene in mouse NIH 3T3 cells induces resistance to cisplatin.

The effect of expression of the c-Ha-ras oncogene on cisplatin (DDP) sensitivity was examined in murine NIH 3T3 cells transfected with the dexamethasone (DEX)-inducible mouse mammary tumor virus promoter linked to an activated c-Ha-ras gene [LTR H-ras(A) cells]. Treatment of these cells with 5 microM DEX for 24 h induced c-Ha-ras expression and produced an 8.2 +/- 1.3-fold (SD) increase in DDP resistance as quantitated by clonogenic assay. Induction of the c-Ha-ras oncogene reduced DDP accumulation by 40% and intrastrand adduct formation by 17%. In nontransfected wild-type NIH 3T3 cells, DEX did not induce DDP resistance nor did it decrease DDP accumulation. Induction of c-Ha-ras expression did not alter cellular glutathione content or the activity of glutathione-S-transferase in the LTR H-ras(A) cells. DEX increased cellular metallothionein content by 1.6-fold in NIH 3T3 cells and 3.3-fold in LTR H-ras(A) cells. We conclude that DEX-induced overexpression of a mutant c-Ha-ras gene confers DDP resistance and that this resistance is associated with an impairment of cellular drug accumulation and an increase in metallothionein content.

Characterization by somatic cell genetics of a monoclonal antibody to the MDR1 gene product (P-glycoprotein): determination of P-glycoprotein expression in multi-drug-resistant KB and CEM cell variants.

We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.

Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye.

Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.

Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein.

Using peroxidase immunohistochemistry, we examined the distribution of P170, a multidrug transport protein, in normal tissues by use of two different monoclonal antibodies (MAb). MAb MRK16 is a MAb that has been shown to react with an epitope in P170 located on the external face of the plasma membrane of multidrug-resistant human cells. MAb C219 has been shown to react with P170 in many mammalian species, and detects an epitope located on the cytoplasmic face of the plasma membrane. Using MRK16, we have previously described the localization of P170 on the bile canalicular face of hepatocytes, the apical surface of proximal tubular cells in kidney, and the surface epithelium in the lower GI tract in normal human tissues. In this work, we report that MRK16 also detects P170 in the capillaries of some human brain samples. A similar pattern was found using MAb C219 in rat tissues. in addition, MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle fibers in rat and human tissues. ATPase cytochemistry showed that these reactive skeletal muscle fibers were of the type I (slow-twitch) class. Other additional sites of C219 reactivity in rat tissues were found in pancreatic acini, seminal vesicle, and testis. Electrophoretic gel immunoblotting showed two protein bands reactive with MAb C219. In liver, MAb C219 reacted with a approximately 170 KD band. In skeletal and cardiac muscle, MAb C219 reacted with a approximately 200 KD band which migrated in the same position as myosin. This band also reacted with an antibody to skeletal muscle myosin. This result suggests that C219 may crossreact with the heavy chain of muscle myosin in cardiac and skeletal muscle. Because MAb C219 reacts with proteins other than P170, it should be used with caution in studies of multidrug resistance.

Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues.

Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDR1), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little P-glycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels of P-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface of cells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.

[Purification of human plasma fibronectin by double affinity chromatography on gelatin and heparin-Trisacryl]. / Purification de la fibronectine plasmatique humaine par double chromatographie d'affinité sur gélatine et héparine-trisacryl.

In this work the human plasma fibronectin was purified by affinity chromatography using a tandem column system. The first affinity column was filled with gelatin-Trisacryl whereas the second one contained heparin-Trisacryl. This double affinity chromatography demonstrated its high efficiency in term of purity and yield. Several analytical methods (electrophoresis, immunoelectrophoresis, F.P.L.C. and adhesion assay on cultured eucaryotic cells) evidenced in fact the high purity of the preparation as well as its biological behaviour in term of cell adhesion and spreading. The performances of the sorbents used facilitate the scaling up when large quantities of FNP are needed.

Spatial visualization of junctional complexes by backscattered electron imaging and silver staining.

A method for the observation of junctional complexes by backscattered electron imaging in scanning electron microscopy, in tissue blocks, is presented. The junctional complexes are revealed by a modified silver staining method (originally devised for nucleolar organizer regions), used "en bloc" after formalin or glutaraldehyde fixation. Backscattered electron imaging allows, after this staining, the observation of junctional complexes through the surface of intact superficial cells, in the three tissues studied (liver, jejunum and urinary bladder). The interest of this approach is to offer the possibility of observing junctional complexes "by transparence," in nondissociated and nonsectioned tissues.

An epidermal proliferative unit-like structure in the epithelium of mouse bladder observed by backscattered electron imaging.

The simultaneous use of a silver-staining technique, backscattered electron imaging and stereo-tilts has made it possible to visualize the spatial distribution of cell nuclei in the stretched epithelium of the bladder of mice. This study has led to the observation that a structural organization resembling the epidermal proliferative unit, previously found in the skin exists also in bladder epithelium. However, the proliferative unit in the bladder was different in that it contained a higher number of cells per unit, and an absence of columns of inactive squamous cells. These findings may indicate that epidermal proliferative unit-like structures represent a common form of organization in some epithelia.

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli.

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the Ag-NOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz-alpha-anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a "rope-like structure". In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripherally, and the weakly staining region and the "rope-like structure" are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.

Improvement in the specificity of the silver staining technique for AgNOR-associated acidic proteins in paraffin sections.

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiff's reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI).

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in "doublets" as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.

Smooth-surfaced control and transformed C3H/10T-1/2 cells differ in cytology: a study by secondary electron, backscattered electron and image analysis.

7,12 dimethylbenz-a-anthracene (DMBA)-treated C3H/10T-1/2 mouse embryo fibroblasts have been characterized by the occurrence of pleomorphic microvilli (PMV) in contrast to the non-treated smooth-surfaced C3H/10T-1/2 cells. Recent studies by semiquantitative secondary electron image (SEI) have demonstrated a significant correlation between the number of cells with numerous PMV and the development of oncogenic potential. In these studies, so-called type III transformants scored highest in oncogenic potential and occurrence of cells with pleomorphic microvilli, but always contained a small fraction of cells displaying rather smooth surfaces. Quantitation of the size of nuclei and nucleoli and the number of intranucleolar silver granules after nucleolar organizer region (AgNOR) staining and back scattered electron image (BEI) with an automatic image analysis system (IBAS) revealed that these smooth-surfaced type III cells were significantly different (p less than 0.001) from the non-DMBA transformed smooth-surfaced control C3H/10T-1/2 cells. Compared to PMV transformants, the smooth transformants exhibited smaller nucleoli, a lesser nucleoli/nuclei fraction and fewer AgNOR sites, indicating reduced cellular activity. The biological significance of the smooth-surfaced fraction in PMV-covered type III transformants remains unclear.