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Environmental stresses adversely affect crop growth and yield, resulting in major losses to plants. These stresses occur simultaneously in nature, and we therefore conducted a meta-analysis in this study to identify differential and shared genes, pathways, and transcriptomic mechanisms involved in Arabidopsis response to biotic and abiotic stresses. The results showed a total of 436/21 significant up-/downregulated differentially expressed genes (DEGs) in response to biotic stresses, while 476 and 71 significant DEGs were respectively up- and downregulated in response to abiotic stresses in Arabidopsis thaliana. In addition, 21 DEGs (2.09%) were commonly regulated in response to biotic and abiotic stresses. Except for WRKY45 and ATXTH22, which were respectively up-/down- and down-/upregulated in response to biotic and abiotic stresses, other common DEGs were upregulated in response to all biotic and abiotic treatments. Moreover, the transcription factors (TFs) bHLH, MYB, and WRKY were the common TFs in response to biotic and abiotic stresses. In addition, ath-miR414 and ath-miR5658 were identified to be commonly expressed in response to both biotic and abiotic stresses. The identified common genes and pathways during biotic and abiotic stresses may provide potential candidate targets for the development of stress resistance breeding programs and for the genetic manipulation of crop plants.

Background Nuclear factors of activated T-cells (NFATs) have been mainly characterized in the context of immune response regulation because, as transcription factors, they have the ability to induce gene transcription. NFAT proteins are found in several types of tumors, for instance, pancreatic carcinoma. The role of NFATs in carcinogenesis is regulating central genes in cell differentiation and cell growth. NFAT proteins are primarily located in cytoplasm and only transported to the cell nucleus after activation. Here, they interact with other transcription factors cooperating with NFAT proteins, thus influencing the selection and regulation of NFAT-controlled genes. To identify and characterize possible interaction partners of the transcription factor NFATc2 in pancreatic carcinoma cells PaTu 8988t. Methods NFATc2 expression and the mode of action of Ionomycin in the pancreatic tumor cell lines PaTu 8988t were shown with Western blotting and immunofluorescence tests. Potential partner proteins were verified by means of immunoprecipitation and binding partners, their physical interactions with DNA pull-down assays, siRNA technologies, and GST pull-down assays. Functional evidence was complemented by reporter–promoter analyses. Results NFATc2 and Sp1 are co-localized in cell nuclei and physically interact at the NFAT target sequence termed NFAT-responsive promotor construct. Sp1 increases the functional activity of its binding partner NFATc2. This interaction is facilitated by Ionomycin in the early stimulation phase (up to 60 min). Conclusions Oncological therapy concepts are becoming more and more specific, aiming at the efficient modulation of specific signal and transcription pathways. The oncogenic transcription partner Sp1 is important for the transcriptional and functional activity of NFATc2 in pancreatic carcinoma. The binding partners interact in cells. Further studies are necessary to identify the underlying mechanisms and establish future therapeutic options for treating this aggressive type of tumor.

Hypertrophy, hyperplasia and altered mucus secretion from the respiratory submucosal glands (SMG) are characteristics of airway diseases such as cystic fibrosis, asthma and chronic bronchitis. More commonly, hyper-secretion of the nasal SMGs contributes to allergic rhinitis and upper airway infection. Considering the role of these glands in disease states, there is a significant dearth in understanding the molecular signals that regulate SMG development and patterning. Due to the imperative role of FGF signalling during the development of other branched structures, we investigated the role of Fgf10 during initiation and branching morphogenesis of murine nasal SMGs. Fgf10 is expressed in the mesenchyme around developing SMGs while expression of its receptor Fgfr2 is seen within glandular epithelial cells. In the Fgf10 null embryo, Steno's gland and the maxillary sinus gland were completely absent while other neighbouring nasal glands showed normal duct elongation but defective branching. Interestingly, the medial nasal glands were present in Fgf10 homozygotes but missing in Fgfr2b mutants, with expression of Fgf7 specifically expressed around these developing glands, indicating that Fgf7 might compensate for loss of Fgf10 in this group of glands. Intriguingly the lateral nasal glands were only mildly affected by loss of FGF signalling, while these glands were missing in Eda mutant mice, where the Steno's and maxillary sinus gland developed as normal. This analysis reveals that regulation of nasal gland development is complex with different subsets of glands being regulated by different signalling pathways. This analysis helps shed light on the nasal gland defects observed in patients with hypohidrotic ectodermal dysplasia (HED) (defect EDA pathway) and LADD syndrome (defect FGFR2b pathway)., Highlights • Murine nasal submucosal glands express Fgf10, Fgfr2 and EDA during embryonic development. • All nasal glands require FGF10 for branching morphogenesis. • FGF10 is required for gland budding and ductal morphogenesis of the Steno's gland and the maxillary sinus glands. • EDA signalling is essential for Lateral Nasal Gland and Medial Nasal Gland budding and ductal morphogenesis.

Plants respond to stresses by producing a broad spectrum of bioactive specialized metabolites. Hormonal elicitors, such as jasmonates, trigger a complex signaling circuit leading to the concerted activation of specific metabolic pathways. However, for many specialized metabolic pathways, the transcription factors involved remain unknown. Here, we report on two homologous jasmonate-inducible transcription factors of the basic helix-loop-helix family, TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (TSAR1) and TSAR2, which direct triterpene saponin biosynthesis in Medicago truncatula. TSAR1 and TSAR2 are coregulated with and transactivate the genes encoding 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE1 (HMGR1) and MAKIBISHI1, the rate-limiting enzyme for triterpene biosynthesis and an E3 ubiquitin ligase that controls HMGR1 levels, respectively. Transactivation is mediated by direct binding of TSARs to the N-box in the promoter of HMGR1. In transient expression assays in tobacco (Nicotiana tabacum) protoplasts, TSAR1 and TSAR2 exhibit different patterns of transactivation of downstream triterpene saponin biosynthetic genes, hinting at distinct functionalities within the regulation of the pathway. Correspondingly, overexpression of TSAR1 or TSAR2 in M. truncatula hairy roots resulted in elevated transcript levels of known triterpene saponin biosynthetic genes and strongly increased the accumulation of triterpene saponins. TSAR2 overexpression specifically boosted hemolytic saponin biosynthesis, whereas TSAR1 overexpression primarily stimulated nonhemolytic soyasaponin biosynthesis. Both TSARs also activated all genes of the precursor mevalonate pathway but did not affect sterol biosynthetic genes, pointing to their specific role as regulators of specialized triterpene metabolism in M. truncatula.

Target gene identification for transcription factors is a prerequisite for the systems wide understanding of organismal behaviour. NAM-ATAF1/2-CUC2 (NAC) transcription factors are amongst the largest transcription factor families in plants, yet limited data exist from unbiased approaches to resolve the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application of complementary functional genomics filters, makes it possible to translate, for each TF, protein binding microarray data into a set of high-quality target genes. With this approach, we confirm NAC target genes reported from independent in vivo analyses. We emphasize that candidate target gene sets together with the workflow associated with functional modules offer a strong resource to unravel the regulatory potential of NAC genes and that this workflow could be used to study other families of transcription factors.

The WRKY family is one of the largest transcription factor (TF) families in plants and plays central roles in modulating plant stress responses and developmental processes, as well as secondary metabolic regulations. Lotus (Nelumbo nucifera) is an aquatic crop that has significant food, ornamental and pharmacological values. Here, we performed an overview analysis of WRKY TF family members in lotus, and studied their functions in environmental adaptation and regulation of lotus benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 65 WRKY genes were identified in the lotus genome and they were well clustered in a similar pattern with their Arabidopsis homologs in seven groups (designated I, IIa-IIe, and III), although no lotus WRKY was clustered in the group IIIa. Most lotus WRKYs were functionally paired, which was attributed to the recently occurred whole genome duplication in lotus. In addition, lotus WRKYs were regulated dramatically by salicilic acid (SA), jasmonic acid (JA), and submergence treatments, and two lotus WRKYs, NnWRKY40a and NnWRKY40b, were significantly induced by JA and promoted lotus BIA biosynthesis through activating BIA biosynthetic genes. The investigation of WRKY TFs for this basal eudicot reveals new insights into the evolution of the WRKY family, and provides fundamental information for their functional studies and lotus breeding.

•Strawberry (Fragaria × ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein).•A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators.•Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3∆). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts.•Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.

MADS-box transcription factors are major regulators of development in flowering plants. The factors act in a combinatorial manner, either as homo- or heterodimers, and they control floral organ formation and identity and many other developmental processes through a complex network of protein¿protein and protein¿DNA interactions. Despite the fact that many studies have been carried out to elucidate MADS-box protein dimerization by yeast systems, very little information is available on the behaviour of these molecules in planta. Here, evidence for specific interactions between the petunia MADS-box proteins FBP2, FBP11, and FBP24 is provided in vivo. The dimers identified in yeast for the ovule-specific FBP24 protein have been confirmed in living plant cells by means of fluorescence resonance energy transfer¿fluorescence lifetime imaging microscopy and, in addition, some of the most likely, less stable homo- and heterodimers were identified. This in vivo approach revealed that particular dimers could only be detected in specific sub-nuclear domains. In addition, evidence for the in planta assembly of these ovule-specific MADS-box transcription factors into higher-order complexes is provided.

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.019) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and 19/20 AML supernatents respectively The degree of irt vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic factors produced by AML cells. The expression of different angiogenic factors was studied using reverse transcription polymerase chain reaction. Cells from 17/20 AML patients showed wide variation in spontaneous vascular endothelial growth factor (VEGF) expression, 4/19 expressed varied spontaneous blastic fibroblast growth factor mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/ or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the growth advantage of the malignant counterpart as a result of the paracrine production of growth factors produced by the surrounding endothelial cells.

Background: MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results: We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions: Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins.

•Strawberry (Fragaria × ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein).•A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators.•Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3∆). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts.•Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.

Plant basic Helix-loop-helix (bHLH) proteins are transcription factors that are involved in many developmental mechanisms, including light signaling and hormone homeostasis. Some of them are non-DNA-binding proteins and could act as dominant negative regulators of other bHLH proteins by forming heterodimers, in a similar way to animal inhibitor of DNA-binding proteins. It has been recently reported that several non-DNA-binding bHLHs are involved in light signaling (KDR/PRE6), gibberellic acid signaling (PRE1/BNQ1/bHLH136) or brassinosteroid signaling (ATBS1). Here we report that Arabidopsis lines overexpressing the PRE3/bHLH135/ATBS1/TMO7 gene are less responsive to red, far-red and blue light than wild-type which is likely to explain the light hyposensitive phenotype displayed when grown under white light conditions. Using quantitative polymerase chain reaction, we show that the expression of PRE3 and KDR/PRE6 genes is regulated by light and that light-related genes are deregulated in the PRE3-ox lines. We show that PRE3 is expressed in the shoot and root meristems and that PRE3-ox lines also have a defect in lateral root development. Our results not only suggest that PRE3 is involved in the regulation of light signaling, but also support the hypothesis that non-DNA-binding bHLH genes are promiscuous genes regulating a wide range of both overlapping and specific regulatory pathways.

Intrahippocampal injection of the endogenous excitotoxin quinolinic acid (QUIN) induces seizures together with local, delayed neurodegeneration in specific cell layers. In situ hybridization histochemistry was used to study the spatio-temporal pattern of expression of neurotrophins (NTFs) after this treatment. As in other excitatory paradigms, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA levels increased dramatically and transiently in dentate gyrus after the administration of 120 nmol of QUIN to the left hippocampus. BDNF, but not NGF, mRNA also increased in the hippocampal pyramidal cell layer, mainly in the CA1 field. Neurotrophin-3 (NT3) mRNA levels decreased in dentate gyrus, practically disappeared around 12 h after the insult and returned to basal levels four days later. A very different pattern of expression of NTFs was found locally: (a) upregulation of NGF and BDNF mRNAs expression was prevented in a spherical region of 1-2 mm diameter around the injection site, (b) a delayed increase in NT3 mRNA levels, beginning at 12 h and lasting for at least 4 days after the administration of QUIN, was found in the same region, in cell layers showing neurodegeneration. Pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg, 30 min before the insult), partially blocked the increase in both BDNF and NGF mRNAs, as well as the decrease in NT3, in the contralateral hippocampus. However, this treatment did not prevent the QUIN-induced local downregulation of NGF and BDNF. Treatment with the AMPA/kainate antagonist NBQX (30 mg/kg, 15 and 5 min before, and 10 min after the insult) did not influence the effect of QUIN upon NGF or BDNF mRNA levels, although it partially prevented the hippocampal contralateral decrease in NT3 mRNA. In conclusion, the present study strongly supports previous work concerning different regulation of BDNF/NGF respect to NT3 in seizure inducing paradigms. Moreover, the different and to some extent opposite regulation of NTFs in the hippocampal region contiguous to the injection site, respect to the remaining hippocampus, suggests a differential regulation of NTFs in QUIN-induced neurodegenerative and seizural processes. Finally, our pharmacological data, (i) show that the upregulation of NGF and BDNF mRNAs, indirectly induced by QUIN, is not mediated by AMPA receptors, and (ii) suggest other effects for QUIN, apart from the stimulation of NMDA receptors.

In this study we have examined the calcium-binding protein expression in rat embryonic (E16) dorsal root ganglia (DRG) neurons in vitro in the presence of neurotrophin-3 (NT-3). A comparison was made with the expression of calcium-binding proteins in DRG subpopulations that depended in vitro on nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF). Our results show that NT-3 promotes the survival of a DRG subpopulation of which over 75% expresses parvalbumin (PV). The majority of these PV-positive NT-3-dependent DRG neurons were large 'type A' neurons. Expression of calbindin-D28k (CaBP) and calretinin (Calr) in the NT-S-dependent DRG population was seen in smaller fractions (between 12 and 17%) of the surviving DRG neurons and in both type A and B neurons. The preferential expression of PV in NT-3-dependent type A neurons is unique in comparison to the expression of PV and the other calcium-binding proteins in DRG neurons surviving in vitro in the presence of NGF or BDNF.

[EN] Structural organization of organs in multicellular organisms occurs through intricate patterning mechanisms that often involve complex interactions between transcription factors in regulatory networks. For example, INDEHISCENT (IND), a basic helix-loop-helix (bHLH) transcription factor, specifies formation of the narrow stripes of valve margin tissue, where Arabidopsis thaliana fruits open on maturity. Another bHLH transcription factor, SPATULA (SPT), is required for reproductive tissue development from carpel margins in the Arabidopsis gynoecium before fertilization. Previous studies have therefore assigned the function of SPT to early gynoecium stages and IND to later fruit stages of reproductive development. Here we report that these two transcription factors interact genetically and via protein-protein contact to mediate both gynoecium development and fruit opening. We show that IND directly and positively regulates the expression of SPT, and that spt mutants have partial defects in valve margin formation. Careful analysis of ind mutant gynoecia revealed slight defects in apical tissue formation, and combining mutations in IND and SPT dramatically enhanced both single-mutant phenotypes. Our data show that SPT and IND at least partially mediate their joint functions in gynoecium and fruit development by controlling auxin distribution and suggest that this occurs through cooperative binding to regulatory sequences in downstream target genes., We thank Steve Penfield for spt-11 and spt-12 mutant seeds, Michael Groszmann for the IND genomic clone, 35S:SPT plants, and for useful discussions, Charlie Scutt for critical comments on the manuscript, Nicolas Arnaud for useful discussions, and Martin F. Yanofsky and Kristina Gremski for sharing unpublished observations and useful discussions. We are grateful to Andrew Davis, Sue Bunnewell, and Kim Findlay at the John Innes Centre for assistance with photography, transmission electron microscopy, and scanning electron microscopy and to Dian Guan for constructing the double G-box vector for the yeast one-hybrid experiment. This work was funded by grants from the Biotechnological and Biological Science Research Council (BB/D018005/1 to L. O.), from the Australian Research Council (A19927094 to D. R. S.), from the Spanish Government (BIO2009-09920 to C. F.), and by a Marie Curie Fellowship (MEST-CT-2005-019727 to S.F.).

This study aimed at defining the role of a basic helix-loop-helix (bHLH) transcription factor gene from Medicago truncatula, MtbHLH1, whose expression is upregulated during the development of root nodules produced upon infection by rhizobia bacteria. We used MtbHLH1 promoter:: GUS fusions and quantitative reverse-transcription polymerase chain reaction analyses to finely characterize the MtbHLH1 expression pattern. We altered MtbHLH1 function by expressing a dominantly repressed construct (CRES-T approach) and looked for possible MtbHLH1 target genes by transcriptomics. We found that MtbHLH1 is expressed in nodule primordia cells derived from pericycle divisions, in nodule vascular bundles (VBs) and in uninfected cells of the nitrogen (N) fixation zone. MtbHLH1 is also expressed in root tips, lateral root primordia cells and root VBs, and induced upon auxin treatment. Altering MtbHLH1 function led to an unusual phenotype, with a modified patterning of nodule VB development and a reduced growth of aerial parts of the plant, even though the nodules were able to fix atmospheric N. Several putative MtbHLH1 regulated genes were identified, including an asparagine synthase and a LOB (lateral organ boundary) transcription factor. Our results suggest that the MtbHLH1 gene is involved in the control of nodule vasculature patterning and nutrient exchanges between nodules and roots.

In order to study the development and functional properties of single, isolated, rat mesencephalic trigeminal neurones, a cell-culture procedure was developed for these specific primary sensory neurones. Mesencephalic trigeminal neurones were isolated from the brainstem of 16-day-old rat embryos. Various factors thought to promote the survival and growth of these neurones in vitro were examined. Outgrowth and maintenance of mesencephalic trigeminal neurones in vitro appeared to be stimulated by a muscle-derived factor, present in muscle-conditioned medium or in muscle extract. Of the neurotrophic factors examined, brain-derived neurotrophic factor and neurotrophin-3, but not nerve-growth factor, promoted the survival of rat mesencephalic trigeminal neurones. Optimal survival of these neurones was found to occur on a monolayer of astrocytes, an effect mediated through direct cell-to-cell interactions.

Neurotrophin-3 has been characterized as the product of a gene cloned by homology with nerve growth factor and brain-derived neurotrophic factor. Recombinant neurotrophin-3, like nerve growth factor and brain-derived neurotrophic factor, has been shown to enhance survival and differentiation of specific neuronal populations in vitro.4,10,15 However, little is known about its function and regulation in vivo. Both brain-derived neurotrophic factor and nerve growth factor messenger RNAs increased in adult rat brain, in a wide range of excitatory paradigms.1,3,11,13,22,28 In contrast, neurotrophin-3 messenger RNA decreased in some of them.13,22 Neurotrophin-3 is the most highly expressed neurotrophic factor in immature areas of the central nervous system. However, no stimulation of its expression in the mature central nervous system, either in physiological or pathological conditions, has been described to date. This behaviour suggests that neurotrophin-3 could be involved in biological roles different from the prototypes nerve growth factor and brain-derived neurotrophic factor. Excitatory amino acid receptor-mediated neurotoxicity (excitotoxicity) is believed to contribute to neuronal loss in a wide range of neurodegenerative conditions (for a review, see Ref. 17). Moreover, locally increased levels of the endogenous excitotoxin quinolinic acid may be involved in the natural development of neurodegenerative diseases.12,23,24 The unilateral intrahippocampal injection of 120 nmol of quinolinic acid induced seizures together with local neurodegeneration in specific cell layers. In situ hybridization histochemistry was used to analyse the spatiotemporal pattern of expression of neurotrophin-3. As in other excitotoxic paradigms, neurotrophin-3 messenger RNA clearly decreased, nearly disappearing, in the contralateral hippocampus. In contrast, a long-lasting specific stimulation of this messenger RNA expression was observed in ipsilateral CA1 and CA4 locally degenerating cell layers. The present results (i) are the first example of an increased expression of neurotrophin-3 messenger RNA in the adult brain, and (ii) suggest the involvement of this neurotrophic factor in the quinolinic acid-mediated neurodegeneration process.

In specific cell types like keratinocytes, Notch signaling plays an important pro-differentiation and tumor suppressing function, with down-modulation of the Notch1 gene being associated with cancer development. Besides being controlled by p53, little else is known on regulation of Notch1 gene expression in this context. We report here that transcription of this gene is driven by a TATA-less “sharp peak” promoter and that the minimal functional region of this promoter, which extends from the −342 bp position to the initiation codon, is differentially active in normal versus cancer cells. This GC rich region lacks p53 binding sites, but binds Klf4 and Sp3. This finding is likely to be of biological significance, as Klf4 and, to a lesser extent, Sp3 are up-regulated in a number of cancer cells where Notch1 expression is down-modulated, and Klf4 over-expression in normal cells is sufficient to down-modulate Notch1 gene transcription. The combined knock-down of Klf4 and Sp3 was necessary for the reverse effect of increasing Notch1 transcription, consistent with the two factors exerting an overlapping repressor function through their binding to the Notch1 promoter.

We have recently identified a gene named GITR (glucocorticoid-induced TNF receptor related gene). GITR is expressed in different cells and tissues such as T lymphocytes from thymus and spleen and lymph nodes, and also in the lung. GITR ligand (GITRL) is expressed in several cells including macrophages, B cells, dendritic cells, and endothelial cells. In the present study, by comparing the responses in wild-type (WT) mice (GITR+/+) and GITR-deficient mice (GITR-/-), we investigated the role played by GITR-GITRL interaction in the development of chronic lung injury caused by bleomycin instillation. When compared with bleomycin-treated GITR+/+ mice, bleomycin-treated GITR-/- mice exhibited a reduced degree of i) lung infiltration with polymorphonuclear neutrophils (MPO activity); ii) edema formation; iii) histological evidence of lung injury; iv) TNF-alpha and interleukin (IL)-1beta production; v) nitrotyrosine formation; and vi) NF-kappaB activation. The cotreatment of GITR+/+ mice with Fc-GITR fusion protein (6.25 microg/mouse) also significantly attenuated all of the above indicators of lung damage and inflammation. Our results clearly demonstrate that GITR-GITRL interaction plays an important role in the chronic lung injury induced by bleomycin in the mice.

All developmental transitions throughout the life cycle of a plant are influenced by light. In Arabidopsis, multiple photoreceptors including the UV-A/blue-sensing cryptochromes (cry1-2) and the red/far-red responsive phytochromes (phyA-E) monitor the ambient light conditions [1 and 2]. Light-regulated protein stability is a major control point of photomorphogenesis [3]. The ubiquitin E3 ligase COP1 (nstitutively hotomorphogenic 1) regulates the stability of several light-signaling components [4, 5 and 6]. HFR1 (long ypocotyl in ar-ed light) is a putative transcription factor with a bHLH domain acting downstream of both phyA and the cryptochromes [7, 8 and 9]. HFR1 is closely related to PIF1, PIF3, and PIF4 (hytochrome nteracting actor 1, 3 and 4), but in contrast to the latter three, there is no evidence for a direct interaction between HFR1 and the phytochromes [7, 10, 11 and 12]. Here, we show that the protein abundance of HFR1 is tightly controlled by light. HFR1 is an unstable phosphoprotein, particularly in the dark. The proteasome and COP1 are required in vivo to degrade phosphorylated HFR1. In addition, HFR1 can interact with COP1, consistent with the idea of COP1 directly mediating HFR1 degradation. We identify a domain, conserved among several bHLH class proteins involved in light signaling [13 and 14], as a determinant of HFR1 stability. Our physiological experiments indicate that the control of HFR1 protein abundance is important for a normal de-etiolation response

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.019) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and 19/20 AML supernatents respectively The degree of irt vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic factors produced by AML cells. The expression of different angiogenic factors was studied using reverse transcription polymerase chain reaction. Cells from 17/20 AML patients showed wide variation in spontaneous vascular endothelial growth factor (VEGF) expression, 4/19 expressed varied spontaneous blastic fibroblast growth factor mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/ or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the growth advantage of the malignant counterpart as a result of the paracrine production of growth factors produced by the surrounding endothelial cells.

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Intrahippocampal injection of the endogenous excitotoxin quinolinic acid (QUIN) induces seizures together with local, delayed neurodegeneration in specific cell layers. In situ hybridization histochemistry was used to study the spatio-temporal pattern of expression of neurotrophins (NTFs) after this treatment. As in other excitatory paradigms, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNA levels increased dramatically and transiently in dentate gyrus after the administration of 120 nmol of QUIN to the left hippocampus. BDNF, but not NGF, mRNA also increased in the hippocampal pyramidal cell layer, mainly in the CA1 field. Neurotrophin-3 (NT3) mRNA levels decreased in dentate gyrus, practically disappeared around 12 h after the insult and returned to basal levels four days later. A very different pattern of expression of NTFs was found locally: (a) upregulation of NGF and BDNF mRNAs expression was prevented in a spherical region of 1-2 mm diameter around the injection site, (b) a delayed increase in NT3 mRNA levels, beginning at 12 h and lasting for at least 4 days after the administration of QUIN, was found in the same region, in cell layers showing neurodegeneration. Pretreatment with the non-competitive NMDA antagonist MK-801 (2 mg/kg, 30 min before the insult), partially blocked the increase in both BDNF and NGF mRNAs, as well as the decrease in NT3, in the contralateral hippocampus. However, this treatment did not prevent the QUIN-induced local downregulation of NGF and BDNF. Treatment with the AMPA/kainate antagonist NBQX (30 mg/kg, 15 and 5 min before, and 10 min after the insult) did not influence the effect of QUIN upon NGF or BDNF mRNA levels, although it partially prevented the hippocampal contralateral decrease in NT3 mRNA. In conclusion, the present study strongly supports previous work concerning different regulation of BDNF/NGF respect to NT3 in seizure inducing paradigms. Moreover, the different and to some extent opposite regulation of NTFs in the hippocampal region contiguous to the injection site, respect to the remaining hippocampus, suggests a differential regulation of NTFs in QUIN-induced neurodegenerative and seizural processes. Finally, our pharmacological data, (i) show that the upregulation of NGF and BDNF mRNAs, indirectly induced by QUIN, is not mediated by AMPA receptors, and (ii) suggest other effects for QUIN, apart from the stimulation of NMDA receptors.

Neurotrophin-3 has been characterized as the product of a gene cloned by homology with nerve growth factor and brain-derived neurotrophic factor. Recombinant neurotrophin-3, like nerve growth factor and brain-derived neurotrophic factor, has been shown to enhance survival and differentiation of specific neuronal populations in vitro.4,10,15 However, little is known about its function and regulation in vivo. Both brain-derived neurotrophic factor and nerve growth factor messenger RNAs increased in adult rat brain, in a wide range of excitatory paradigms.1,3,11,13,22,28 In contrast, neurotrophin-3 messenger RNA decreased in some of them.13,22 Neurotrophin-3 is the most highly expressed neurotrophic factor in immature areas of the central nervous system. However, no stimulation of its expression in the mature central nervous system, either in physiological or pathological conditions, has been described to date. This behaviour suggests that neurotrophin-3 could be involved in biological roles different from the prototypes nerve growth factor and brain-derived neurotrophic factor. Excitatory amino acid receptor-mediated neurotoxicity (excitotoxicity) is believed to contribute to neuronal loss in a wide range of neurodegenerative conditions (for a review, see Ref. 17). Moreover, locally increased levels of the endogenous excitotoxin quinolinic acid may be involved in the natural development of neurodegenerative diseases.12,23,24 The unilateral intrahippocampal injection of 120 nmol of quinolinic acid induced seizures together with local neurodegeneration in specific cell layers. In situ hybridization histochemistry was used to analyse the spatiotemporal pattern of expression of neurotrophin-3. As in other excitotoxic paradigms, neurotrophin-3 messenger RNA clearly decreased, nearly disappearing, in the contralateral hippocampus. In contrast, a long-lasting specific stimulation of this messenger RNA expression was observed in ipsilateral CA1 and CA4 locally degenerating cell layers. The present results (i) are the first example of an increased expression of neurotrophin-3 messenger RNA in the adult brain, and (ii) suggest the involvement of this neurotrophic factor in the quinolinic acid-mediated neurodegeneration process.