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Direct Target (direct + target)
Selected AbstractsJarid2 is among a set of genes differentially regulated by Nkx2.5 during outflow tract morphogenesisDEVELOPMENTAL DYNAMICS, Issue 7 2010Jeremy L. Barth Abstract Nkx2.5, a transcription factor implicated in human congenital heart disease, is required for regulation of second heart field (SHF) progenitors contributing to outflow tract (OFT). Here, we define a set of genes (Lrrn1, Elovl2, Safb, Slc39a6, Khdrbs1, Hoxb4, Fez1, Ccdc117, Jarid2, Nrcam, and Enpp3) expressed in SHF containing pharyngeal arch tissue whose regulation is dependent on Nkx2.5. Further investigation shows that Jarid2, which has been implicated in OFT morphogenesis, is a direct target of Nkx2.5 regulation. Jarid2 expression was up-regulated in SHF mesoderm of Nkx2.5-deficient embryos. Chromatin immunoprecipitation analysis showed Nkx2.5 interaction with consensus binding sites in the Jarid2 promoter in pharyngeal arch cells. Finally, Jarid2 promoter activity and mRNA expression levels were down-regulated by Nkx2.5 overexpression. Given the role of Jarid2 as a regulator of early cardiac proliferation, these findings highlight Jarid2 as one of several potential mediators of the critical role played by Nkx2.5 during OFT morphogenesis. Developmental Dynamics 239:2024,2033, 2010. © 2010 Wiley-Liss, Inc. [source] Fjx1: A notch-inducible secreted ligand with specific binding sites in developing mouse embryos and adult brainDEVELOPMENTAL DYNAMICS, Issue 3 2005Rebecca Rock Abstract The mouse fjx1 gene was identified as a homologue to the Drosophila gene four-jointed (fj). Fj encodes a transmembrane type II glycoprotein that is partially secreted. The gene was found to be a downstream target of the Notch signaling pathway in leg segmentation and planar cell polarity processes during eye development of Drosophila. Here, we show that fjx1 is not only conserved in vertebrates, but we also identified the murine fjx1 gene as a direct target of Notch signaling. In addition to the previously described expression of fjx1 in mouse brain, we show here that fjx1 is expressed in the peripheral nervous system, epithelial cells of multiple organs, and during limb development. The protein is processed and secreted as a presumptive ligand. Through the use of an fjx1-AP fusion protein, we could visualize fjx1 binding sites at complementary locations, supporting the notion that fjx1 may function as a novel signaling molecule. Developmental Dynamics 234:602,612, 2005. © 2005 Wiley-Liss, Inc. [source] Overexpression of inducible nitric oxide synthase and accumulation of 8-OHdG in nasopharyngeal carcinomaHISTOPATHOLOGY, Issue 2 2008Y Segawa Aims:, Nitric oxide (NO), produced by inducible NO synthase (iNOS), has been suggested to cause oxidative stress, leading to 8-hydroxydeoxyguanosine (8-OHdG) accumulation and subsequent transversion mutation of DNA. The aim was to evaluate iNOS expression and the status of oxidative stress in nasopharyngeal carcinoma (NPC). Methods and results:, Seventy-three cases of NPC were investigated to examine the immunohistochemical expression of iNOS, 8-OHdG and latent membrane protein-1 (LMP-1) and Epstein,Barr virus-encoded small RNA (EBER) expression using in situ hybridization. iNOS mRNA expression and p53 gene mutations were also assessed. Overexpression of iNOS, LMP-1 and EBER was observed in 62 (84.9%), 28 (38.4%) and 53 (72.6%) cases respectively. p53 gene mutation was found in 10 of 73 (13.7%) cases. Immunohistochemical iNOS expression was associated with the 8-OHdG labelling index, iNOS mRNA expression and p53 gene alteration (P < 0.0001, P = 0.016 and 0.0082 respectively). Conclusions:, Our present findings suggest that the expression of iNOS induces oxidative stress in NPC. Although the presence of p53 mutation was associated with iNOS overexpression, the type of acid,base change of p53 was transition, but not transversion, which suggests that the p53 gene is not the direct target of DNA damage by 8-OHdG accumulation. [source] The functions of freezing in the social interactions of juvenile high- and low-aggressive miceAGGRESSIVE BEHAVIOR, Issue 6 2001Daniel J. Bauer Abstract Selectively bred low-aggressive mice are frequently observed to freeze on social contact, despite the fact that this behavior was never a direct target of selection. To elucidate this finding, the present research aimed to identify the possible functions freezing may serve in social interactions. It was hypothesized that freezing may modify social interactions through self-regulatory mechanisms and/or via its modulating effects on the actions of social partners. These hypotheses were evaluated with respect to the sequential changes observed over the course of a 10-min dyadic test in freezing, social reactivity, and approaches among juvenile (24,30-day-old) mice from the NC900 and NC100 high- and low-aggressive lines. Analyses of the patterns of social interactions between subjects and partners revealed two primary results. First, freezing was more than an expression of fear; it also functioned as a regulator of emotional arousal, as suggested by the substantial reduction of reactive behaviors seen in animals that showed high levels of freezing. Second, freezing functioned to facilitate high levels of affiliative social interaction with social partners. The implications of these results for understanding how the differentiation of the NC900 and NC100 occurred within microevolution and development are discussed. Aggr. Behav. 27:463,475, 2001. © 2001 Wiley-Liss, Inc. [source] Effect of miRNA-10b in regulating cellular steatosis level by targeting PPAR-, expression, a novel mechanism for the pathogenesis of NAFLDJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 1 2010Lin Zheng Abstract Background and Aim:, Accumulating evidence supports the effects of miRNA in lipid metabolism, providing a potential linkage between certain miRNA and non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the miRNA expression pattern in a steatotic L02 cell model and explore the function of certain miRNA target pairs. Methods:, The cell model was established by culturing L02 cells with a high concentration of free fatty acid. Micro-array and stem-loop reverse transcription polymerase chain reaction (RT,PCR) were utilized to detect dysregulated miRNA, whereas computational algorithms were used for target prediction. Real time RT,PCR, Western blot, luciferase activity measurement, and other techniques were employed for target verification. Results:, Seventeen upregulated and 15 downregulated miRNA were found in steatotic L02 cells, while miRNA-10b was proven to regulate the steatosis level. Peroxisome proliferator-activated receptor-, (PPAR-,) was also found to participate in steatosis, as its protein level was decreased in steatotic L02 cells and its overexpression by transfection into the PPAR-,,pcDNA 3.1 vector could partially alleviate steatosis. We further found that PPAR-, is the direct target of miRNA-10b as it showed significantly changed protein expression, but a relatively unchanged mRNA level in steatotic L02 cells transfected with pre-miRNA-10b and anti-miRNA-10b. Moreover, the action of miRNA-10b on PPAR-, depends on the presence of a single miRNA-10b binding site, as the activity of a luciferase reporter carrying the mutant PPAR-, 3,-untranslated region was not reduced by the expression of miRNA-10b. Conclusion:, The established miRNA profile of the steatotic L02 cell model and the novel effect of miRNA-10b in regulating hepatocyte steatosis may provide a new explanation of the pathogenesis of NAFLD. [source] Direct Inhibitory Effect of Glucocorticoids on Corticotrophin-Releasing Hormone Gene Expression in Neurones of the Paraventricular Nucleus in Rat Hypothalamic Organotypic CulturesJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2008B. Bali Corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory neurones of hypothalamic paraventricular nucleus governs neuroendocrine stress cascade and is the major target of the negative feedback effect of corticosteroids. To assess whether glucocorticoids exert their inhibitory effect on CRH expression directly on parvocellular neurones or indirectly through a complex neuronal circuit, we examined the effect of corticosterone (CORT) and dexamethasone (DEX) on CRH mRNA levels in slice explant cultures of the rat hypothalamus. Organotypic slice cultures were prepared from 6 days old rat pups and maintained in vitro for 14 days. CRH mRNA expression was measured by in situ hybridisation histochemistry. Under basal conditions, CRH mRNA expressing cells were exclusively revealed in the paraventricular region along the third ventricle. Inhibition of action potential spike activity by tetrodotoxin (TTX, 1 ,m) reduced CRH mRNA signal in the organotypic cultures. CORT (500 nm) or DEX (50 nm) treatment for 24 h significantly inhibited CRH expression in the parvocellular neurones and this effect of corticosteroids was not affected following blockade of voltage dependent sodium channels by TTX. Forskolin-stimulated CRH mRNA levels in the paraventricular nucleus were also inhibited by CORT or DEX in the presence and in the absence of TTX. These studies identify paraventricular CRH neurones as direct target of corticosteroid feedback. Type II corticosteroid receptor agonists act directly on paraventricular neurones to inhibit basal and forskolin-induced CRH mRNA expression in explant cultures of the rat hypothalamus. [source] Regulatory factor X4 variant 3: A transcription factor involved in brain development and disease,JOURNAL OF NEUROSCIENCE RESEARCH, Issue 16 2007Donghui Zhang Abstract Regulatory factor X4 variant 3 (RFX4_v3) is a recently identified transcription factor specifically expressed in the brain. Gene disruption in mice demonstrated that interruption of a single allele (heterozygous, +/,) prevented formation of the subcommissural organ (SCO), resulting in congenital hydrocephalus, whereas interruption of two alleles (homozygous, ,/,) caused fatal failure of dorsal midline brain structure formation. These mutagenesis studies implicated RFX4_v3 in early brain development as well as the genesis of the SCO. Rfx4_v3 deficiency presumably causes abnormalities in brain by altering the expression levels of many genes that are crucial for brain morphogenesis, such as the signaling components in the Wnt, bone morphogenetic protein, and retinoic acid pathways. RFX4_v3 might affect these critical signaling pathways in brain development. Cx3cl1, a chemokine gene highly expressed in brain, was identified as a direct target for RFX4_v3, indicating that RFX4_v3 possesses trans -acting activity to stimulate gene expression. Rfx4_v3 is highly expressed in the suprachiasmatic nucleus and might be involved in regulating the circadian clock. One haplotype in RFX4_v3 gene is linked to a higher risk of bipolar disorder, suggesting that this protein might contribute to the pathogenesis of the disease. This Mini-Review describes our current knowledge about RFX4_v3, an important protein that appears to be involved in many aspects of brain development and disease. © 2007 Wiley-Liss, Inc. [source] Significance of consensus CYC-binding sites found in the promoters of both ChCYC and ChRAD genes in Chirita heterotricha (Gesneriaceae)JOURNAL OF SYSTEMATICS EVOLUTION, Issue 4 2010Xia YANG Abstract,CYC -like genes are widely conserved in controlling floral dorsoventral asymmetry (zygomorphy) through persistent expression in corresponding domains in core eudicots. To understand how CYC -like gene expression is maintained during flower development, we selected Chirita heterotricha as a material and isolated the promoter sequences of the ChCYC1C and ChCYC1D genes, homologs of CYC, by inverse polymerase chain reaction. Further promoter analyses led to the identification of a putative cis -regulatory element in each promoter matching the consensus DNA binding site for Antirrhinum CYC protein: GGCCCCTC at ,165 for ChCYC1C, and GGCCCCCC at ,163 for ChCYC1D. This indicates that both the ChCYC1C and ChCYC1D genes have probably evolved autoregulatory loops to sustain their expression in developing flowers. We also isolated the coding and promoter sequences of the ChRAD gene, a homolog of Antirrhinum RAD. Promoter analysis showed that the ChRAD gene promoter also contained a putative CYC-binding site (GGCCCAC at ,134). Therefore, ChRAD is likely a direct target of the ChCYC1 genes, which is similar to Antirrhinum RAD. These results imply that the establishment of floral zygomorphy in Chirita may have been achieved by the evolution of an autoregulatory loop for CYC -like genes, which was probably accompanied by simultaneous co-option of the RAD -like gene into their regulatory network. [source] Early Hepatic Microvascular Injury in Response to Acetaminophen ToxicityMICROCIRCULATION, Issue 5 2003YOSHIYA ITO ABSTRACT Objective: The hepatic toxic response to acetaminophen (APAP) is characterized by centrilobular (CL) necrosis preceded by hepatic microvascular injury and congestion. The present study was conducted to examine changes in liver microcirculation after APAP dosing. Methods: Male C57Bl/6 mice were treated with APAP (600 mg/kg body weight) by oral gavage. The livers of anesthetized mice were examined using established in vivo microscopic methods at 0, 0.5, 1, 2, 4, 6, 12 hours after APAP. Results: The levels of hepatic transaminases (i.e., alanine aminotransferase [ALT] and aspartate transaminase) increased minimally for up to 2 hours. Thereafter, their levels were significantly and progressively increased. The numbers of swollen sinusoidal endothelial cells (SECs) in periportal regions were increased (3.5-fold) from 0.5 to 6 hours, and those in CL regions were increased (4.0-fold) at 0.5 and 1 hour. The intensity of in vivo staining for formaldehyde-treated serum albumin, which is a specific ligand for SECs, was reduced from 2 to 12 hours. Erythrocytes infiltrated into the space of Disse as early as 2 hours, and the area occupied by these cells was markedly increased at 6 hours. Sinusoidal perfusion was reduced from 1 through 12 hours, with a nadir (35% decrease) at 4 and 6 hours. Phagocytic Kupffer cell activity was significantly elevated from 0.5 through 12 hours. Although gadolinium chloride minimized the changes in sinusoidal blood flow and reduced ALT levels 6 hours after APAP, it failed to inhibit endothelial swelling, extravasation of erythrocytes, and CL parenchymal necrosis. Conclusions: These results confirm that APAP-induced SEC injury precedes hepatocellular injury, supporting the hypothesis that SECs are an early and direct target for APAP toxicity. These findings also suggest that reduced sinusoidal perfusion and increased Kupffer cell activity contribute to the development of APAP-induced liver injury. [source] Crystal structure of Mycobacterium tuberculosis LrpA, a leucine-responsive global regulator associated with starvation responsePROTEIN SCIENCE, Issue 1 2008Manchi C.M. Reddy Abstract The bacterial leucine-responsive regulatory protein (Lrp) is a global transcriptional regulator that controls the expression of many genes during starvation and the transition to stationary phase. The Mycobacterium tuberculosis gene Rv3291c encodes a 150-amino acid protein (designated here as Mtb LrpA) with homology with Escherichia coli Lrp. The crystal structure of the native form of Mtb LrpA was solved at 2.1 Å. The Mtb LrpA structure shows an N-terminal DNA-binding domain with a helix-turn-helix (HTH) motif, and a C-terminal regulatory domain. In comparison to the complex of E. coli AsnC with asparagine, the effector-binding pocket (including loop 100,106) in LrpA appears to be largely preserved, with hydrophobic substitutions consistent with its specificity for leucine. The effector-binding pocket is formed at the interface between adjacent dimers, with an opening to the core of the octamer as in AsnC, and an additional substrate-access channel opening to the outer surface of the octamer. Using electrophoretic mobility shift assays, purified Mtb LrpA protein was shown to form a protein,DNA complex with the lat promoter, demonstrating that the lat operon is a direct target of LrpA. Using computational analysis, a putative motif is identified in this region that is also present upstream of other operons differentially regulated under starvation. This study provides insights into the potential role of LrpA as a global regulator in the transition of M. tuberculosis to a persistent state. [source] Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 7 2010Amandine Mullier The median eminence is one of the seven so-called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood-borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we report on our use of immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO-1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO-1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties. J. Comp. Neurol. 518:943,962, 2010. © 2009 Wiley-Liss, Inc. [source] Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 7 2010Amandine Mullier Abstract The median eminence is one of the seven so-called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood-borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we utilize immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO-1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO-1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties. J. Comp. Neurol. 518:943,962, 2010. © 2009 Wiley-Liss, Inc. [source] Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene productTHE PLANT JOURNAL, Issue 5 2000Wim Reidt Summary The Arabidopsis mutants fus3 and abi3 show pleiotropic effects during embryogenesis including reduced levels of transcripts encoding embryo-specific seed proteins. To investigate the interaction between the B3-domain-containing transcription factors FUS3 and ABI3 with the RY cis -motif, conserved in many seed-specific promoters, a promoter analysis as well as band-shift experiments were performed. The analysis of promoter mutants revealed the structural requirements for the function of the RY cis -element. It is shown that both the nucleotide sequence and the alternation of purin and pyrimidin nucleotides (RY character) are essential for the activity of the motif. Further, it was shown that FUS3 and ABI3 can act independently of each other in controlling promoter activity and that the RY cis -motif is a target for both transcription factors. For FUS3, which is so far the smallest known member of the B3-domain family, a physical interaction with the RY motif was established. The functional and biochemical data demonstrate that the regulators FUS3 and ABI3 are essential components of a regulatory network acting in concert through the RY-promoter element to control gene expression during late embryogenesis and seed development. [source] Mutational and expressional analysis of BNIP3, a pro-apoptotic Bcl-2 member, in gastric carcinomas,APMIS, Issue 11 2007SUNG HAK LEE Cell death deregulation is a hallmark of human cancers. BNIP3 was initially identified as a pro-apoptotic member of the Bcl-2 family and plays an important role in apoptosis, necrosis and autophagy. The aim of this study was to see whether alterations of BNIP3 protein expression and somatic mutation of the BNIP3 gene are characteristics of human cancers. We analyzed the expression of BNIP3 protein in 60 gastric adenocarcinomas by immunohistochemistry. In addition, we analyzed BNIP3 mutation in the DNA sequences encoding BH3 (Bcl-2 homology3) and TM (transmembrane) domains that are important in the cell death function of BNIP3 by single-strand conformation polymorphism (SSCP) in 48 colorectal, 48 gastric, and 48 breast carcinomas, and 48 acute leukemias. By immunohistochemistry, BNIP3 protein was detected in 40 of the 60 carcinomas (67%). Both early and advanced gastric carcinomas expressed BNIP3. There was no significant association between BNIP3 expression and clinicopathologic characteristics, including invasion, metastasis and stage. In contrast to the cancer cells, epithelial cells in normal gastric mucosa showed no or weak expression of BNIP3. Mutational analysis revealed BNIP3 mutation in neither the BH3 nor the TM domain, suggesting that BNIP3 mutation in these domains is not a direct target of inactivation in gastric, colorectal and breast carcinomas, and acute leukemias. Increased expression of BNIP3 in the malignant gastric epithelial cells compared to the normal mucosal epithelial cells suggests that BNIP3 expression might play a role in gastric carcinoma development. [source] Immunohistochemical analysis of phospho-BAD protein and mutational analysis of BAD gene in gastric carcinomas,APMIS, Issue 8 2007EUN GOO JEONG Mounting evidence indicates that deregulation of apoptosis contributes to the development of human cancers. BAD, a proapoptotic Bcl-2 family protein, regulates the intrinsic apoptosis pathway. The aim of this study was to explore whether alterations of phospho-BAD (p-BAD) protein that antagonizes apoptosis function of BAD and mutation of BAD gene are characteristics of human gastric cancers. We analyzed expression of p-BAD in 60 gastric adenocarcinomas by immunohistochemistry. Also, we analyzed BAD gene for detection of somatic mutations by single-strand conformation polymorphism (SSCP) assay. p-BAD expression was detected well in normal gastric mucosal epithelial cells, whereas it was detected in only 51% (31 of the 60) of the cancers. There was no somatic mutation of BAD gene in the 60 gastric cancer samples. The decreased expression of p-BAD in malignant gastric epithelial cells compared to normal mucosal epithelial cells suggested that loss of p-BAD expression may play a role in gastric tumorigenesis. The data also suggest that BAD mutation may not be a direct target of inactivation in gastric tumorigenesis. [source] The fitness advantage of a high-performance weaponBIOLOGICAL JOURNAL OF THE LINNEAN SOCIETY, Issue 4 2009JERRY F. HUSAK Weapons used in combat between males are usually attributed to sexual selection, which operates via a fitness advantage for males with weapons of better ,quality'. Because the performance capacity of morphological traits is typically considered the direct target of selection, Darwin's intrasexual selection hypothesis can be modified to predict that variation in reproductive success should be explained by variation in performance traits relevant to combat. Despite such a straightforward prediction, tests of this hypothesis are conspicuously lacking. We show that territorial male collared lizards with greater bite-force capacity sire more offspring than weaker biting rivals but exhibit no survival advantage. We did not detect stabilizing or disruptive selection on bite-force capacity. Taken together, these results support the hypothesis that superior weapon performance provides a fitness advantage through increased success in male contests. Sexual selection on weapon performance therefore appears to be a force driving the evolution and maintenance of sexual dimorphism in head shape. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 96, 840,845. [source] A Computational Study of Feedback Effects on Signal Dynamics in a Mitogen-Activated Protein Kinase (MAPK) Pathway ModelBIOTECHNOLOGY PROGRESS, Issue 2 2001Anand R. Asthagiri Exploiting signaling pathways for the purpose of controlling cell function entails identifying and manipulating the information content of intracellular signals. As in the case of the ubiquitously expressed, eukaryotic mitogen-activated protein kinase (MAPK) signaling pathway, this information content partly resides in the signals' dynamical properties. Here, we utilize a mathematical model to examine mechanisms that govern MAPK pathway dynamics, particularly the role of putative negative feedback mechanisms in generating complete signal adaptation, a term referring to the reset of a signal to prestimulation levels. In addition to yielding adaptation of its direct target, feedback mechanisms implemented in our model also indirectly assist in the adaptation of signaling components downstream of the target under certain conditions. In fact, model predictions identify conditions yielding ultra-desensitization of signals in which complete adaptation of target and downstream signals culminates even while stimulus recognition (i.e., receptor-ligand binding) continues to increase. Moreover, the rate at which signal decays can follow first-order kinetics with respect to signal intensity, so that signal adaptation is achieved in the same amount of time regardless of signal intensity or ligand dose. All of these features are consistent with experimental findings recently obtained for the Chinese hamster ovary (CHO) cell lines (Asthagiri et al., J. Biol. Chem.1999, 274, 27119,27127). Our model further predicts that although downstream effects are independent of whether an enzyme or adaptor protein is targeted by negative feedback, adaptor-targeted feedback can "back-propagate" effects upstream of the target, specifically resulting in increased steady-state upstream signal. Consequently, where these upstream components serve as nodes within a signaling network, feedback can transfer signaling through these nodes into alternate pathways, thereby promoting the sort of signaling cross-talk that is becoming more widely appreciated. [source] Oligonucleotide-based microarray analysis of retinoic acid target genes in the protochordate, Ciona intestinalisDEVELOPMENTAL DYNAMICS, Issue 4 2005Tomoko Ishibashi Abstract Oligonucleotide-based microarray analyses were carried out to identify retinoic acid target genes in embryos of the ascidian Ciona intestinalis. Of 21,938 spots, 50 (corresponding to 43 genes) showed over twofold up-regulation in retinoic acid-treated tail bud embryos. In situ hybridization verified retinoic acid-induced up-regulation of 23 genes. Many of them were expressed in the anterior tail region, where a retinaldehyde dehydrogenase homolog is expressed. Homologs of vertebrate genes involved in neurogenesis and/or neuronal functions (e.g., COUP-TF, Ci-Hox1, and SCO-spondin) were expressed in the central nervous system of Ciona embryos, and activated by retinoic acid. Genes encoding transcription factors (e.g., Ci-lmx1.2, vitamin D receptor, and Hox proteins) and apoptosis-related proteins (e.g., transglutaminase and an apoptosis-inducing factor homolog) were also activated by retinoic acid. Simultaneous treatment of embryos with retinoic acid and puromycin revealed a few direct targets, including genes encoding Ci-Hox1, Ci-Cyp26, and an Rnf126-like ring finger protein. Developmental Dynamics 233:1571,1578, 2005. © 2005 Wiley-Liss, Inc. [source] Nitric oxide regulates axonal regeneration in an insect embryonic CNSDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2008Michael Stern Abstract In higher vertebrates, the central nervous system (CNS) is unable to regenerate after injury, at least partially because of growth-inhibiting factors. Invertebrates lack many of these negative regulators, allowing us to study the positive factors in isolation. One possible molecular player in neuronal regeneration is the nitric oxide (NO),cyclic guanosine-monophosphate (cGMP) transduction pathway which is known to regulate axonal growth and neural migration. Here, we present an experimental model in which we study the effect of NO on CNS regeneration in flat-fillet locust embryo preparations in culture after crushing the connectives between abdominal ganglia. Using whole-mount immunofluorescence, we examine the morphology of identified serotonergic neurons, which send a total of four axons through these connectives. After injury, these axons grow out again and reach the neighboring ganglion within 4 days in culture. We quantify the number of regenerating axons within this period and test the effect of drugs that interfere with NO action. Application of exogenous NO or cGMP promotes axonal regeneration, whereas scavenging NO or inhibition of soluble guanylyl cyclase delays regeneration, an effect that can be rescued by application of external cGMP. NO-induced cGMP immunostaining confirms the serotonergic neurons as direct targets for NO. Putative sources of NO are resolved using the NADPH-diaphorase technique. We conclude that NO/cGMP promotes outgrowth of regenerating axons in an insect embryo, and that such embryo-culture systems are useful tools for studying CNS regeneration. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2008 [source] EXPERIMENTAL MANIPULATION OF SEXUAL SELECTION PROMOTES GREATER MALE MATING CAPACITY BUT DOES NOT ALTER SPERM INVESTMENTEVOLUTION, Issue 4 2009Helen S. Crudgington Sexual selection theory makes clear predictions regarding male spermatogenic investment. To test these predictions we used experimental sexual selection in Drosophila pseudoobscura, a sperm heteromorphic species in which males produce both fertile and sterile sperm, the latter of which may function in postmating competition. Specifically, we determined whether the number and size of both sperm types, as well as relative testis mass and accessory gland size, increased with increased sperm competition risk and whether any fitness benefits could accrue from such changes. We found no effect of sexual selection history on either the number or size of either sperm morph, or on relative testis mass. However, males experiencing a greater opportunity for sexual selection evolved the largest accessory glands, had the greatest mating capacity, and sired the most progeny. These findings suggest that sterile sperm are not direct targets of sexual selection and that accessory gland size, rather than testis mass, appears to be an important determinant of male reproductive success. We briefly review the data from experimental sexual selection studies and find that testis mass may not be a frequent target of postcopulatory sexual selection and, even when it is, the resulting changes do not always improve fitness. [source] Synergistic induction of cyclin D1 in oligodendrocyte progenitor cells by IGF-I and FGF-2 requires differential stimulation of multiple signaling pathwaysGLIA, Issue 10 2007Terra J. Frederick Abstract D-type cyclins are direct targets of extracellular signals and critical regulators of G1 progression. Our previous data demonstrated that IGF-I and FGF-2 synergize to enhance cyclin D1 expression, cyclin E/cdk2 complex activation, and S-phase entry in OP cells. Here, we provide a mechanistic explanation for how two growth factor signaling pathways converge on a major cell cycle regulator. IGF-I and FGF-2 differentially activate signaling pathways to coordinately promote cyclin D1 expression. We show that the p44/p42 MAPK signaling pathway is essential for FGF-2 induction of cyclin D1 mRNA. In contrast, blocking the PI3-Kinase pathway results in loss of IGF-I/FGF-2 synergistic induction of cyclin D1 protein levels. Moreover, the presence of IGF-I significantly enhances nuclear localization of cyclin D1, which also requires PI3K signaling. GSK-3,, a downstream target of the PI3K/Akt pathway, is phosphorylated in the presence of IGF-I in OPs. Consistent with a known role for GSK-3, in cyclin D1 degradation, we show that proteasome inhibition in OPs exposed to FGF-2 increased cyclin D1 levels, equivalent to levels seen in IGF-I/FGF-2 treated cells. Thus, we provide a model for cyclin D1 coordinate regulation where FGF-2 stimulation of the MAPK pathway promotes cyclin D1 mRNA expression while IGF-I activation of the PI3K pathway inhibits proteasome degradation of cyclin D1 and enhances nuclear localization of cyclin D1. © 2007 Wiley-Liss, Inc. [source] MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells,HEPATOLOGY, Issue 1 2009Teng Xu Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down-regulation of miR-195 has been observed in various types of cancers. However, the biological function of miR-195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR-195. Our results showed that miR-195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR-195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR-195 blocked G1/S transition, whereas inhibition of miR-195 promoted cell cycle progression. Subsequent investigation characterized multiple G1/S transition-related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR-195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR-195, whereas overexpression of these proteins attenuated miR-195-induced G1 arrest. In addition, miR-195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR-195 may block the G1/S transition by repressing Rb-E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR-195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR-195 in cancer therapy. (HEPATOLOGY 2009.) [source] p53 may positively regulate hepatocyte proliferation in ratsHEPATOLOGY, Issue 2 2002Yukiko Inoue p53, known as a tumor suppressor gene, is a transcription factor that regulates various cellular functions. Recently, several growth factor gene promoters, including that of transforming growth factor , (TGF-,), were shown to be direct targets of p53-mediated transcription. Hepatic p53 mRNA is up-regulated during liver regeneration in rats. The aim of this study is to examine the role of p53 in hepatocyte proliferation. p53 protein levels were examined in rat hepatocytes cultured in the medium containing hepatocyte growth factor (HGF). p53 levels began to increase after 6 hours of incubation, reached a maximum at 18 hours, and decreased thereafter. DNA synthesis increased at 12 hours and peaked at 30 hours. When hepatocytes were incubated with p53 antisense oligonucleotide in addition to HGF, increases of p53 and TGF-, levels were suppressed, and DNA synthesis was reduced. The increases of TGF-, levels and DNA synthesis were also suppressed by a chemical inhibitor of p53, pifithrin-,. In rats after two-thirds partial hepatectomy, hepatic p53 increased and reached maximal levels around 16 hours when hepatic HGF levels have been shown to reach a maximum followed by an increase in hepatic TGF-, levels or hepatocyte proliferation. In contrast, sham-operated rats showed minor elevations of hepatic p53 levels. In conclusion, p53 production is stimulated by HGF and may contribute to the proliferation of rat hepatocytes. Considering previous findings indicating the importance of endogenous TGF-, for the proliferation of hepatocytes stimulated by HGF, TGF-, might play a role in HGF-p53 mediated hepatocyte proliferation. [source] HIV-1 neuropathogenesis: glial mechanisms revealed through substance abuseJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Kurt F. Hauser Abstract Neuronal dysfunction and degeneration are ultimately responsible for the neurocognitive impairment and dementia manifest in neuroAIDS. Despite overt neuronal pathology, HIV-1 does not directly infect neurons; rather, neuronal dysfunction or death is largely an indirect consequence of disrupted glial function and the cellular and viral toxins released by infected glia. A role for glia in HIV-1 neuropathogenesis is revealed in experimental and clinical studies examining substance abuse,HIV-1 interactions. Current evidence suggests that glia are direct targets of substance abuse and that glia contribute markedly to the accelerated neurodegeneration seen with substance abuse in HIV-1 infected individuals. Moreover, maladaptive neuroplastic responses to chronic drug abuse might create a latent susceptibility to CNS disorders such as HIV-1. In this review, we consider astroglial and microglial interactions and dysfunction in the pathogenesis of HIV-1 infection and examine how drug actions in glia contribute to neuroAIDS. [source] Molecular targets for the cancer preventive activity of tea polyphenolsMOLECULAR CARCINOGENESIS, Issue 6 2006Chung S. Yang Abstract Inhibition of carcinogenesis by tea and tea polyphenols has been demonstrated in many animal models. The mechanisms of action have been extensively investigated mostly in cell culture systems with (-)-epigallocatechin-3-gallate (EGCG), the most active and major polyphenolic compound from green tea. However, the mechanisms of cancer preventive activity by tea and tea polyphenols are not clearly understood. This article discusses some of the reported mechanisms and possible targets for the action of EGCG. The difficulties and major issues in extrapolating data from studies in cancer cell lines to cancer prevention mechanisms are discussed. Activities observed in cell culture with high concentrations of EGCG may not be relevant because of the limited systemic bioavailability of EGCG. In addition, possible artifacts due to the auto-oxidation of EGCG may complicate this issue. Some recent studies revealed high-affinity EGCG binding proteins as possible direct targets for the action of EGCG. Validating the related cancer preventive mechanisms found in in vitro studies in animal models and human samples would be exciting. © 2006 Wiley-Liss, Inc. [source] The RNA binding protein CsrA controls cyclic di-GMP metabolism by directly regulating the expression of GGDEF proteinsMOLECULAR MICROBIOLOGY, Issue 1 2008Kristina Jonas Summary The carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobility shift assays confirmed the direct and specific binding of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH resulted in a more than 20-fold increase in the cellular concentration of the second messenger cyclic di-GMP (c-di-GMP), implying that both proteins possess diguanylate cyclase activity. Phenotypic characterization revealed that both proteins are involved in the regulation of motility in a c-di-GMP-dependent manner. CsrA was also found to regulate the expression of five additional GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels of c-di-GMP. All together, these data demonstrate a global role for CsrA in the regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins at the post-transcriptional level. [source] | ||||||||||||||||||||||||||||