The flavonoid naringenin stimulates the intercellular colonization of wheat roots by Azorhizobium caulinodans

We have studied intercellular colonization of wheat roots by Azorhizobium caulinodans and other diazotrophic bacteria, using strains marked with the lacZ reporter gene to facilitate their detection and identification. A. caulinodans was observed by light and electron microscopy to enter the roots of wheat at high frequency at the points of emergence of lateral roots (lateral root cracks). After lateral root crack colonization, bacteria moved into intercellular spaces within the cortical cell layer of roots. The flavonoid naringenin at 10 and 100 mmol m^–3 significantly stimulated root colonization. The roles of the structural nodABC genes and the regulatory nodD gene were also studied; lateral root crack colonization of wheat was shown to be Nod factor- and NodD-independent. Similar frequencies of lateral root crack colonization were observed following inoculation of wheat with Azospirillum brasilense. Colonization by A. brasilense was stimulated by naringenin and also by other flavonoid molecules.     

ONIOM DFT/PM3 calculation on the interaction between STI-571 and abelson tyrosine kinase

The ONIOM2 (B3LYP/6–31G (d, p): PM3) and B3LYP/6–31G (d, p) methods were applied to investigate the interaction between STI-571 and abelson tyrosine kinase binding site. The complex of N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)- phenyl]-benzamide (part of STI-571) and related 16 amino acid residues were found at B3LYP/6–31G (d, p) level to have hydrogen bonds and π....π stacking interaction, their binding energy via HAF optimization was −20.4 kcal mol⁻¹. The results derived from this study agreed well with the reported observation. Figure Optimized structure of STI-571 and Thr315 in abelson tyrosine kinase based on ONIOM2 method     

茎瘤固氮根瘤菌Azorhizobium caulinodans ORS571基因组分泌蛋白的生物信息学分析

为了获取茎瘤固氮根瘤菌(Azorhizobium caulinodans ORS571)的分泌蛋白,以便更深入地了解该菌的共生固氮作用,本研究采用SignalP、TMHMM、PSORTb、TargetP、LipoP、TatP和SecretomeP软件对该菌全部4717个蛋白序列进行分析预测。结果共识别了653个分泌蛋白,其中具有分泌型信号肽的蛋白54个,具有RR-motif型信号肽的蛋白1个,具有脂蛋白信号肽的蛋白2个和非经典分泌蛋白596个。该菌含信号肽分泌蛋白仅占全部蛋白的1.2%,低于其它固氮菌。在分泌蛋白中识别了核酸内切酶和核糖核酸酶等6个核酸酶。它们可能参与宿主植物遗传物质的降解,干扰宿主遗传代谢,进一步在宿主植物侵染过程中起到重要作用。此外还识别了超氧化物歧化酶、过氧化氢酶和谷胱甘肽S-转移酶等4个抗氧化酶。它们可能参与活性氧的清除以保护固氮酶,是该菌固氮过程的重要参与者。     

毛萼田菁茎瘤菌被种子浸提物诱导表达基因的筛选及其功能

摘要：【目的】选择毛萼田菁茎瘤菌ORS571为研究对象,筛选毛萼田菁茎瘤菌在菌植互作早期被宿主植物种子浸提物诱导表达的基因。【方法】采用新颖的基于抗性的活体表达筛选技术,以种子浸提物为诱导物,筛选获得能够稳定被种子浸提物诱导的突变株。通过中间片断融合报告基因的方法研究信号分子对相关基因的诱导情况。【结果】随机引物PCR产物测序及网上BLAST结果表明,被诱导表达的基因为LysE家族的成员。而且,对另外3个属根瘤菌中lysE基因的研究发现,它们都可以被相应的宿主植物种子浸提物诱导表达。此外,这四株根瘤菌的lysE突变株都表现对刀豆氨酸极为敏感。本文首次报道了lysE家族在根瘤菌中的生理功能。【结论】我们推测：LysE家族蛋白可能广泛存在于根瘤菌中,并在根瘤菌与宿主植物的早期相互作用中发挥重要作用。     

Cisplatin-evoked DNA fragmentation in normal and cancer cells and its modulation by free radical scavengers and the tyrosine kinase inhibitor STI571

Cis-diamminedichloroplatinum(II) (cisplatin, cis-DDP) is well studied anticancer drug, whose activity can be attributed to its ability to form adducts with DNA, but this drug can also form DNA-damaging free radicals, however this mechanism of cisplatin action is far less explored. Using the comet assay we studied cisplatin-induced DNA damage in the presence of spin traps: DMPO and PBN, Vitamins A, C and E as well as the tyrosine kinases inhibitor STI571 in normal human lymphocytes and leukemic K562 cells. The latter cells express the BCR/ABL fusion protein, which can be a target of the tyrosine kinase inhibitor STI571. A 20 h incubation with cisplatin at 1-10 microM induced DNA cross-links and DNA fragmentation in normal and cancer cells. Cisplatin could induce intra- and interstrand DNA-DNA cross-links as well as DNA-protein cross-links. DNA damage in K562 cells was more pronounced than in normal lymphocytes. In the presence of spin traps and vitamins we noticed a decrease in the DNA fragmentation in both cell types. Co-treatment of the lymphocytes with cisplatin at 10 microM and STI571 at 0.25 microg/ml caused an increase of DNA fragmentation in comparison with DNA fragmentation induced by cisplatin alone. In the case of K562 cells, an increase of DNA fragmentation was observed after treatment with cisplatin at 1 microM. Our results indicate that the free radicals scavengers could decrease DNA fragmentation induced by cisplatin in the normal and cancer cells, but probably they have no effect on DNA cross-linking induced by the drug. The results obtained with the BCR/ABL inhibitor suggest that K562 cells could be more sensitive towards co-treatment of cisplatin and STI571. Our results suggest also that aside from the BCR/ABL other factors such as p53 level, signal transduction pathways and DNA repair processes can be responsible for the increased sensitivity of K562 cells to cisplatin compared with normal lymphocytes.     

STI571 reduces TRAIL-induced apoptosis in colon cancer cells: c-Abl activation by the death receptor leads to stress kinase-dependent cell death

Background

In an effort to achieve better cancer therapies, we elucidated the combination cancer therapy of STI571 (an inhibitor of Bcr-Abl and clinically used for chronic myelogenous leukemia) and TNF-related apoptosis-inducing ligand (TRAIL, a developing antitumor agent) in leukemia, colon, and prostate cancer cells.

Methods

Colon cancer (HCT116, SW480), prostate cancer (PC3, LNCaP) and leukemia (K562) cells were treated with STI571 and TRAIL. Cell viability was determined by MTT assay and sub-G1 appearance. Protein expression and kinase phosphorylation were determined by Western blotting. c-Abl and p73 activities were inhibited by target-specific small interfering (si)RNA. In vitro kinase assay of c-Abl was conducted using CRK as a substrate.

Results

We found that STI571 exerts opposite effects on the antitumor activity of TRAIL. It enhanced cytotoxicity in TRAIL-treated K562 leukemia cells and reduced TRAIL-induced apoptosis in HCT116 and SW480 colon cancer cells, while having no effect on PC3 and LNCaP cells. In colon and prostate cancer cells, TRAIL caused c-Abl cleavage to the active form via a caspase pathway. Interestingly, JNK and p38 MAPK inhibitors effectively blocked TRAIL-induced toxicity in the colon, but not in prostate cancer cells. Next, we found that STI571 could attenuate TRAIL-induced c-Abl, JNK and p38 activation in HCT116 cells. In addition, siRNA targeting knockdown of c-Abl and p73 also reduced TRAIL-induced cytotoxicity, rendering HCT116 cells less responsive to stress kinase activation, and masking the cytoprotective effect of STI571.

Conclusions

All together we demonstrate a novel mediator role of p73 in activating the stress kinases p38 and JNK in the classical apoptotic pathway of TRAIL. TRAIL via caspase-dependent action can sequentially activate c-Abl, p73, and stress kinases, which contribute to apoptosis in colon cancer cells. Through the inhibition of c-Abl-mediated apoptotic p73 signaling, STI571 reduces the antitumor activity of TRAIL in colon cancer cells. Our results raise additional concerns when developing combination cancer therapy with TRAIL and STI571 in the future.     

Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [¹⁸F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [¹³¹I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [¹⁸F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [¹⁸F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.     

Pine wood nematode (PWN)-secretory antigen 571 as a biomarker for the PWN and its monoclonal antibodies for detecting PWN and its infected pine tree

The biochemical characteristics of pine wood nematode (PWN)-secretory antigen 571 (PWN-SA571) identified from the PWN secretome and PWN-infected pine tree extracts (PWNIPT) were investigated. PWN-SA571 has homologs in various nematodes in diverse families and a signal peptide at the amino-terminal end. Since it was not possible to express the full-length PWN-SA571 protein, two peptides showing high antigenicity were synthesized and used as antigens for generating monoclonal antibodies (Mabs). Mab-secreting fusion hybridoma cell lines were selected based on immunoreactivity to free peptide and BSA-conjugated peptide (BSA pep) antigens by enzyme-linked immunosorbent assay for establishing Mab-secreting hybridoma lines. Ascites containing PWN-SA571-specific Mabs showed stronger immunoreactivity to PWNIPT than to healthy pine tree extracts. In addition, PWN-SA571-specific Mabs could recognize less than 20 ng of BSA pep in lateral flow assays. Furthermore, purified biotin-conjugated PWN-SA571-specific Mab IgG or IgM were able to detect BSA pep (<15 ng) and PWN extracts (<600 ng). Taken together, these results demonstrate that PWN-SA571-specific Mabs could detect PWN or PWNIPT extracts by ELISA, LFA, or dot blot analysis.     

STI571 (Glivec) affects histamine release and intracellular pH after alkalinisation in HMC-1560, 816

The human mast cell line (HMC-1(560, 816)) was used to study the effect of the tyrosine kinase inhibitor STI571 (Glivec) on exocytosis, intracellular Ca(2+) and pH changes, because STI571 inhibits the proliferation of HMC-1(560) and induces its apoptosis. This drug does not have these effects on HMC-1(560, 816). Exocytosis in HMC-1(560, 816) cells can be stimulated by alkalinisation with NH(4)Cl as well as with ionomycin. Surprisingly 24-h pre-incubation with STI571 decreases spontaneous histamine release of HMC-1(560, 816) cells, but increases the histamine response after alkalinisation and not after ionomycin-stimulation. After addition of NH(4)Cl, pH(i) has a higher increase in STI571 pre-incubated cells, without changing intracellular Ca(2+) concentration. Activation of PKC in combination with tyrosine kinase inhibition increases also histamine release in HMC-1(560, 816) cells. Strangely, STI571 pre-incubated cells with PKC inhibited by rottlerin show the same effects. In these cells, cytosolic pH increases more than in control cells. This is the first report of STI571 effect in HMC-1(560, 816) cells. It seems that different pathways modulate signals for proliferation and exocytosis. STI571 does not only inhibit KIT TyrK, but may also influence cytosolic pH after alkalinisation in both cell lines, HMC-1(560) and HMC-1(560, 816), and this ends in induced histamine release. This work is important since HMC-1(560, 816) cells are reported in 80% of aggressive systemic mastocytosis cases and the understanding of some signalling pathways involved in mast cell response could facilitate drug targeting.