A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.

Pro-apoptotic gene expression mediated by the p38 mitogen-activated protein kinase signal transduction pathway

Neurotrophic factor deprivation causes apoptosis by a mechanism that requires macromolecular synthesis. This fact suggests that gene expression is necessary to achieve cell death. To identify mRNA that is expressed in apoptotic cells we used subtractive hybridization with cDNA prepared from neuronal pheochromocytoma cells. Monoamine oxidase (MAO) expression was increased in cells during nerve growth factor withdrawal-induced apoptosis. The increased apoptosis and induction of MAO was prevented by inhibition of the p38 mitogen-activated protein (MAP) kinase pathway. MAO may contribute to the apoptotic process because inhibition of MAO activity suppressed cell death. Together, these data indicate that MAO may be a target of pro-apoptotic signal transduction by the p38 MAP kinase pathway.

Platelets modulate gastric ulcer healing: Role of endostatin and vascular endothelial growth factor release

Bleeding and delayed healing of ulcers are well recognized clinical problems associated with the use of aspirin and other nonsteroidal antiinflammatory drugs, which have been attributed to their antiaggregatory effects on platelets. We hypothesized that antiplatelet drugs might interfere with gastric ulcer healing by suppressing the release of growth factors, such as vascular endothelial growth factor (VEGF), from platelets. Gastric ulcers were induced in rats by serosal application of acetic acid. Daily oral treatment with vehicle, aspirin, or ticlopidine (an ADP receptor antagonist) was started 3 days later and continued for 1 week. Ulcer induction resulted in a significant increase in serum levels of VEGF and a significant decrease in serum levels of endostatin (an antiangiogenic factor). Although both aspirin and ticlopidine markedly suppressed platelet aggregation, only ticlopidine impaired gastric ulcer healing and angiogenesis as well as reversing the ulcer-associated changes in serum levels of VEGF and endostatin. The effects of ticlopidine on ulcer healing and angiogenesis were mimicked by immunodepletion of circulating platelets, and ticlopidine did not influence ulcer healing when given to thrombocytopenic rats. Incubation of human umbilical vein endothelial cells with serum from ticlopidine-treated rats significantly reduced proliferation and increased apoptosis, effects reversed by an antibody directed against endostatin. Ticlopidine treatment resulted in increased platelet endostatin content and release. These results demonstrate a previously unrecognized contribution of platelets to the regulation of gastric ulcer healing. Such effects likely are mediated through the release from platelets of endostatin and possibly VEGF. As shown with ticlopidine, drugs that influence gastric ulcer healing may do so in part through altering the ability of platelets to release growth factors.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-β

Epithelial–mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-β (TGF-β) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-β-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-β within 4 hours after treatment. TGF-β-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell–matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-β. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-γ when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type β1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-γ. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr51 release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-β1 or anti-V-β2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-β can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type β1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8+ cytolytic T cells.

Induction of cyclooxygenase-2 by Epstein–Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IκBα(S32A/S36A), which is not phosphorylated and prevents NF-κB activation, with LMP1 showed that NF-κB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-κB is involved in LMP1-induced cox-2 promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-κB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E2 in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato1

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.

Acid-Growth Response and α-Expansins in Suspension Cultures of Bright Yellow 2 Tobacco1

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of α-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple α-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

The Phytochrome Response of the Lemna gibba NPR1 Gene Is Mediated Primarily through Changes in Abscisic Acid Levels1

Two important signaling systems involved in the growth and development of plants, those triggered by the photoreceptor phytochrome and the hormone abscisic acid (ABA), are involved in the regulation of expression of the NPR1 gene of Lemna gibba. We previously demonstrated that phytochrome action mediates changes in ABA levels in L. gibba, correlating with changes in gene expression evoked by stimulation of the phytochrome system. We have now further characterized phytochrome- and ABA-mediated regulation of L. gibba NPR1 gene expression using a transient particle bombardment assay, demonstrating that regulatory elements controlling responses to both stimuli reside within 156 nucleotides upstream of the transcription start. Linker scan (LS) analysis of the region from −156 to −70 was used to identify two specific requisite and nonredundant cis-acting promoter elements between −143 to −135 (LS2) and −113 to −101 (LS5). Mutation of either of these elements resulted in a coordinate loss of regulation by phytochrome and ABA. This suggests that, unlike the L. gibba Lhcb2*1 promoter, in which phytochrome and ABA regulatory elements are separable, the phytochrome response of the L. gibba NPR1 gene can be attributed to alterations in ABA levels.

The rise and fall of the medical mediated searcher

The relationship between the development of mediated online literature searching and the recruitment of medical librarians to fill positions as online searchers was investigated. The history of database searching by medical librarians was outlined and a content analysis of thirty-five years of job advertisements in MLA News from 1961 through 1996 was summarized. Advertisements for online searchers were examined to test the hypothesis that the growth of mediated online searching was reflected in the recruitment of librarians to fill positions as mediated online searchers in medical libraries. The advent of end-user searching was also traced to determine how this trend affected the demand for mediated online searching and job availability of online searchers. Job advertisements were analyzed to determine what skills were in demand as end-user searching replaced mediated online searching as the norm in medical libraries. Finally, the trend away from mediated online searching to support of other library services was placed in the context of new roles for medical librarians.

Critical role for the docking-protein FRS2α in FGF receptor-mediated signal transduction pathways

The docking protein FRS2α has been implicated as a mediator of signaling via fibroblast growth factor receptors (FGFRs). We have demonstrated that targeted disruption of FRS2α gene causes severe impairment in mouse development resulting in embryonal lethality at E7.0–E7.5. Experiments with FRS2α-deficient fibroblasts demonstrate that FRS2α plays a critical role in FGF-induced mitogen-activated protein (MAP) kinase stimulation, phosphatidylinositol-3 (PI-3) kinase activation, chemotactic response, and cell proliferation. Following FGF stimulation, tyrosine phosphorylated FRS2α functions as a site for coordinated assembly of a multiprotein complex that includes Gab1 and the effector proteins that are recruited by this docking protein. Furthermore, we demonstrate that different tyrosine phosphorylation sites on FRS2α are responsible for mediating different FGF-induced biological responses. These experiments establish the central role of FRS2α in signaling via FGFRs and demonstrate that FRS2α mediates multiple FGFR-dependent signaling pathways critical for embryonic development.

Activation of NF-κB by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK–IKKα/β–IκBα and MKK3/6–p38 MAP kinase signaling pathways in epithelial cells

Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation of NF-κB, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here, we show that NTHi strongly activates NF-κB in human epithelial cells via two distinct signaling pathways, NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves activation of NF-κB inducing kinase (NIK)–IKKα/β complex leading to IκBα phosphorylation and degradation, whereas the NF-κB translocation-independent pathway involves activation of MKK3/6–p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase pathways may occur at transforming growth factor-β activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor 2 (TLR2) is required for NTHi-induced NF-κB activation. In addition, several key inflammatory mediators including IL-1β, IL-8, and tumor necrosis factor-α are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane of all NTHi and H. influenzae type b strains, appears to also activate NF-κB via similar signaling pathways. Taken together, our results demonstrate that NTHi activates NF-κB via TLR2–TAK1-dependent NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase signaling pathways. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.

CrkII signals from epidermal growth factor receptor to Ras.

A rat fibroblast mutant defective in oncogenic transformation and signaling from epidermal growth factor receptor to Ras has been isolated. The mutant contains dominant negative-type point mutations in the C-terminal SH3 domain of one crkII gene. Among the adapters tested, the mutant is complemented only by crkII cDNA. Expression of the mutated crkII in parent cells generates the phenotype indistinguishable from the mutant cell. Yet overexpression or reduced expression of Grb2 in the mutant before and after complementation with crkII have little effect on its phenotype. We conclude that adapter molecules are highly specific and that the oncogenic growth signal from epidermal growth factor receptor to Ras is predominantly mediated by CrkII in rat fibroblast.