Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.

Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway.

Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 beta-stimulated mesangial cells: evidence for the involvement of transcriptional and posttranscriptional regulation.

Inducible nitric oxide synthase (iNOS; EC 1.14.13.39) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin 1 beta (IL-1 beta). We have reported that nanomolar concentrations of dexamethasone suppress IL-1 beta-induced iNOS protein expression and production of nitrite, the stable end product of NO formation, without affecting IL-1 beta-triggered increase in iNOS mRNA levels. We now have studied the mechanisms by which dexamethasone suppresses IL-1 beta-stimulated iNOS expression in mesangial cells. Surprisingly, nuclear run-on transcription experiments demonstrate that dexamethasone markedly attenuates IL-1 beta-induced iNOS gene transcription. However, this is counteracted by a prolongation of the half-life of iNOS mRNA from 1 h to 2.5 h by dexamethasone. Moreover, dexamethasone drastically reduces the amount of iNOS protein by reduction of iNOS mRNA translation and increased degradation of iNOS protein. These results indicate that glucocorticoids act at multiple levels to regulate iNOS expression, thus providing important insights into the treatment of inflammatory diseases.

Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides.

Pituitary follicle-stimulating hormone (FSH) induces CREM gene expression in Sertoli cells: involvement in long-term desensitization of the FSH receptor.

Transcription factor CREM (cAMP-responsive element modulator) plays a pivotal role in the nuclear response to cAMP in neuroendocrine cells. We have previously shown that follicle-stimulating hormone (FSH) directs CREM expression in male germ cells. The physiological importance of FSH in Sertoli cell function prompted us to analyze its effect on CREM expression in these cells. We observed a dramatic and specific increase in the CREM isoform ICER (inducible cAMP early repressor) expression, with a peak 4 h after FSH treatment of primary Sertoli cells. Interestingly, induced levels of ICER protein persist for a considerably longer time. Induction of the repressor ICER accompanies early down-regulation of the FSH receptor transcript, which leads to long-term desensitization. Here we show that ICER represses FSH receptor expression by binding to a CRE-like sequence in the regulatory region of the gene. Our results confirm the crucial role played by CREM in hormonal control and suggest its role in the long-term desensitization phenomenon of peptide membrane receptors.

Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation.

In Alzheimer disease (AD) the amyloid beta-peptide (A beta) accumulates in plaques in the brain. A beta can be neurotoxic by a mechanism involving induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels ([Ca2+]i). In light of evidence for an inflammatory response in the brain in AD and reports of increased levels of tumor necrosis factor (TNF) in AD brain we tested the hypothesis that TNFs affect neuronal vulnerability to A beta. A beta-(25-35) and A beta-(1-40) induced neuronal degeneration in a concentration- and time-dependent manner. Pretreatment of cultures for 24 hr with TNF-beta or TNF-alpha resulted in significant attenuation of A beta-induced neuronal degeneration. Accumulation of peroxides induced in neurons by A beta was significantly attenuated in TNF-pretreated cultures, and TNFs protected neurons against iron toxicity, suggesting that TNFs induce antioxidant pathways. The [Ca2+]i response to glutamate (quantified by fura-2 imaging) was markedly potentiated in neurons exposed to A beta, and this action of A beta was suppressed in cultures pretreated with TNFs. Electrophoretic mobility-shift assays demonstrated an induction of a kappa beta-binding activity in hippocampal cells exposed to TNFs. Exposure of cultures to I kappa B (MAD3) antisense oligonucleotides, a manipulation designed to induce NF-kappa B, mimicked the protection by TNFs. These data suggest that TNFs protect hippocampal neurons against A beta toxicity by suppressing accumulation of ROS and Ca2+ and that kappa B-dependent transcription is sufficient to mediate these effects. A modulatory role for TNF in the neurodegenerative process in AD is proposed.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.

Tumor necrosis factor (TNF) is selectively cytotoxic to some types of tumor cells in vitro and exerts antitumor activity in vivo. Reactive oxygen intermediates (ROIs) have been implicated in the direct cytotoxic activity of TNF. By using confocal microscopy, flow cytometry, and the ROI-specific probe dihydrorhodamine 123, we directly demonstrate that intracellular ROIs are formed after TNF stimulation. These ROIs are observed exclusively under conditions where cells are sensitive to the cytotoxic activity of TNF, suggesting a direct link between both phenomena. ROI scavengers, such as butylated hydroxyanisole, effectively blocked the formation of free radicals and arrested the cytotoxic response, confirming that the observed ROIs are cytocidal. The mitochondrial glutathione system scavenges the major part of the produced ROIs, an activity that could be blocked by diethyl maleate; under these conditions, TNF-induced ROIs detectable by dihydrorhodamine 123 oxidation were 5- to 20-fold higher.

Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only.

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

Involvement of the ALL-1 gene in a solid tumor.

Translocations involving chromosome band 11q23, found in 5-10% of human acute leukemias, disrupt the ALL-1 gene. This gene is fused by reciprocal translocation with a variety of other genes in acute lymphoblastic and myelogenous leukemias, and it undergoes self-fusion in acute myeloid leukemias with normal karyotype or trisomy 11. Here we report an alteration of the ALL-1 gene in a gastric carcinoma cell line (Mgc80-3). Characterization of this rearrangement revealed a three-way complex translocation, involving chromosomes 1 and 11, resulting in a partial duplication of the ALL-1 gene. Sequencing of reverse transcription-PCR products and Northern blot analysis showed that only the partially duplicated ALL-1 gene was transcribed, producing an mRNA with exon 8 fused to exon 2. This report of ALL-1 gene rearrangement in a solid tumor suggests that ALL-1 plays a role in the pathogenesis of some solid malignancies. The absence of the normal transcript in this cell line, in association with the loss-of-heterozygosity studies on chromosome 11q23 seen in solid tumors, suggests that ALL-1 is involved in tumorigenesis by a loss-of-function mechanism.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

Latino Family Involvement: An Exploratory Study of Latina Mother-Daughter Relationships and their Effects on Educational Attainment and Resiliency

Latino family involvement is an important issue in the field of education. Effective strategies to promote family involvement in the Latino community are vital for the educational attainment of Latino students and emotional wellbeing of Latino families. This study used focus groups, in-depth interviews, and observations to examine Latino family involvement and the relationships and communication patterns between Latina mothers and daughters. The Latina mother-daughter relationship was studied in an effort to gain a better understanding of how this relationship affects a Latina daughter's educational attainment and sense of resiliency. Results indicated that a positive relationship between a Latina mother and daughter can increase a Latina daughter's level of educational attainment and sense of resiliency. Additionally, a Latina daughter's level of self-motivation can affect her level of educational attainment as well. Cultural narratives were found to be a common type of communication pattern used between Latina mothers and daughters. They were used to teach cultural values, life lessons, and experiential learning. By improving family involvement efforts within the Latino culture, Latino students will likely see drastic improvements in their overall levels of educational attainment and emotional wellbeing in schools. Implications for Latino students and families, schools that work with Latino families, and educational policy are also discussed.

Involvement of loop 5 lysine residues and the N-terminal β-hairpin of the ribotoxin hirsutellin A on its insecticidal activity

Ribotoxins are cytotoxic members of the family of fungal extracellular ribonucleases best represented by RNase T1. They share a high degree of sequence identity and a common structural fold, including the geometric arrangement of their active sites. However, ribotoxins are larger,with a well-defined N-terminal β-hairpin, and display longer and positively charged unstructured loops. These structural differences account for their cytotoxic properties.Unexpectedly, the discovery of hirsutellin A (HtA), a ribotoxin produced by the invertebrate pathogen Hirsutella thompsonii, showed how it was possible to accommodate these features into a shorter amino acid sequence. Examination of HtA N-terminal β-hairpin reveals differences in terms of length, charge, and spatial distribution. Consequently,four different HtA mutants were prepared and characterized. One of them was the result of deleting this hairpin [Δ(8-15)] while the other three affected single Lys residues in its close spatial proximity (K115E, K118E, and K123E). The results obtained support the general conclusion that HtA active site would show a high degree of plasticity,being able to accommodate electrostatic and structural changes not suitable for the other previously known larger ribotoxins, as the variants described here only presented small differences in terms of ribonucleolytic activity and cytotoxicity against cultured insect cells.