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mRNA Encoding (mrna + encoding)
Selected AbstractsmRNA Encoding a Putative RNA Helicase of the DEAD-Box Gene Family is Up-Regulated in Trypomastigotes of Trypanosoma cruziTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2000ALBERTO M. DÍAZ AÑEL ABSTRACT. Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands. [source] c-Src kinase activation regulates preprotachykinin gene expression and substance P secretion in rat sensory gangliaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2003Orisa J. Igwe Abstract Increased synthesis of substance P (SP) in the dorsal root ganglia (DRG) and enhanced axonal transport to and secretion from the primary afferent sensory neurons might enhance pain signalling in the spinal dorsal horn by modifying pronociceptive pathways. IL-1, increases SP synthesis by enhancing the expression of preprotachykinin (PPT) mRNA encoding for SP and other tachykinins in the DRG. Stimulation of IL-1 receptor by IL-1, may induce the phosphorylation of tyrosine residues in many effector proteins through the activation of p60c-src kinase. The hypothesis that the synthesis of SP in and secretion from the primary sensory ganglia are regulated by the activation of p60c-src kinase induced by IL-1, was tested. Pretreatment of DRG neurons in culture with herbimycin A, genistein or PP2, three structurally different nonreceptor tyrosine kinase inhibitors that act by different mechanisms, decreased the kinase activity of p60c-src induced by the activation of IL-1 receptor. PP3, a negative control for the Src family of tyrosine kinase inhibitor PP2 had no effect. Herbimycin A and genistein also decreased IL-1,-induced expression of PPT mRNA-encoding transcripts and the levels of SP-li synthesized in the cells and secreted into the culture medium in a concentration-dependent manner. SB 203580 [a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor] and PD 98059 (a p44/42 MAPK kinase inhibitor) were ineffective in modulating IL-1,-induced SP synthesis and secretion, and p60c-src kinase activity in DRG neurons. Whereas, IL-1 receptor antagonist and cycloheximide inhibited IL-1,-evoked secretion of SP-like immunoreactivity (SP-li), actinomycin D decreased it significantly but did not entirely abolish it. These findings show that phosphorylation of specific protein tyrosine residue(s) following IL-1 receptor activation might play a key role in IL-1, signalling to modulate PPT gene expression and SP secretion in sensory neurons. In view of the role of SP as an immunomodulator, these studies provide a new insight into neural-immune intercommunication in pain regulation in the sensory ganglia through the IL-1,-induced p60c-src activation. [source] Bone morphogenetic protein-7 enhances dendritic growth and receptivity to innervation in cultured hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2000G. S. Withers Abstract Members of the bone morphogenetic protein (BMP) family of growth factors are present in the central nervous system during development and throughout life. They are known to play an important regulatory role in cell differentiation, but their function in postmitotic telencephalic neurons has not been investigated. To address this question, we examined cultured hippocampal neurons following treatment with bone morphogenetic protein-7 (BMP-7, also referred to as osteogenic protein-1). When added at the time of plating, BMP-7 markedly stimulated the rate of dendritic development. Within 1 day, the dendritic length of BMP-7-treated neurons was more than twice that of controls. By three days the dendritic arbors of BMP-7-treated neurons had attained a level of branching similar to that of 2-week-old neurons cultured under standard conditions. Several findings indicate that BMP-7 selectively enhances dendritic development. While dendritic length was significantly increased in BMP-7-treated neurons, the length of the axon was not. In addition, the mRNA encoding the dendritic protein MAP2 was significantly increased by BMP-7 treatment, but the mRNA for tubulin was not. Finally, BMP-7 did not enhance cell survival. Because dendritic maturation is a rate-limiting step in synapse formation in hippocampal cultures, we examined whether BMP-7 accelerated the rate at which neurons became receptive to innervation. Using two separate experimental paradigms, we found that the rate of synapse formation (assessed by counting synapsin I-positive presynaptic vesicle clusters) was increased significantly in neurons that had been exposed previously to BMP-7. Because BMP-7 and related BMPs are expressed in the hippocampus in situ, these factors may play a role in regulating dendritic branching and synapse formation in both development and plasticity. [source] The initiator caspase, caspase-10,, and the BH-3-only molecule, Bid, demonstrate evolutionary conservation in Xenopus of their pro-apoptotic activities in the extrinsic and intrinsic pathwaysGENES TO CELLS, Issue 7 2006Katsuya Kominami Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10,), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10, and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10, and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10, mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development. [source] The effect of elevated oocyte triiodothyronine content on development of rainbow trout embryos and expression of mRNA encoding for thyroid hormone receptorsJOURNAL OF FISH BIOLOGY, Issue 1 2004J. C. Raine The ability of developing rainbow trout Oncorhynchus mykiss embryos to compensate for elevated oocyte triiodothyronine (T3) content and whether elevation of oocyte T3 content within a physiologically meaningful range affects growth rates of the embryo or the expression of genes encoding for thyroid hormone receptors ,(TR,) and ,(TR,) were examined. Oocytes were immersed in ovarian fluid alone (control) or T3 -enriched ovarian fluid prior to fertilization and water hardening, to induce a dose-dependant increase in oocyte T3 content of c. 3 (control), c. 30 (LT3) or c. 110 ng egg,1(HT3). To examine the interaction of embryo somatic growth with altered thyroid state more effectively, the embryos were reared at two ambient temperatures (8·5 and 5·5°C ) to induce different growth rates. A significant decline in whole embryo T3 content was measured in the T3 -treatment groups reared at both water temperatures by 3 weeks post-fertilization (dpf), and may have reflected the action of outer ring monodeiodinase, which was present in microsomes prepared from embryos 23 dpf. Whole embryo T3 levels in the HT3 group, however, remained higher than controls until phase 2 of development [the onset of endogenous thyroid hormone (TH) release]. This suggested that the embryos exerted some control over their response to exogenous TH, but that there was a limit to the level of control exerted by the embryonic tissues. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of mRNA encoding for the two TR isoforms as early as 26 dpf, and quantitative real-time RT-PCR (qPCR) was used to examine the effect of elevated oocyte T3 content on the expression of these TR genes in embryos raised at 8·5 and 5·5° C, and sampled at similar developmental stages prior to the onset of embryonic TH synthesis, to ensure that the oocyte T3 was the only source of TH exposure to the embryo. There was a suppression of the TR, gene expression in the control 5·5° C group relative to the control 8·5° C group. In addition, both TR, and TR, mRNA accumulation was lower, relative to the controls, in the LT3 treatment group reared at 8·5° C suggesting a suppressive effect of the lower level of T3 treatment on the TR gene expression. Conversely, there were no differences from controls in the HT3 treatment group, possibly indicating that this level of exposure overrides the down-regulating capacity of the embryo. Similar patterns were seen for TR, and TR, mRNA accumulation in embryos reared at 5·5° C, but because of the temperature suppressed level of TR, mRNA in the controls, significant affects of the LT3 treatment were only found for TR,. There were no measurable effects of T3 treatment on oocyte fertility or embryo somatic growth for either temperature treatment group, nor was somatic growth hormone content (measured only in the 8·5° C treatment group) apparently related to in ovo T3 levels. The results suggest that altered in ovo T3 levels, within the ranges used here, do not induce marked affects on embryo development, probably because of the ability of the embryo to maintain the integrity of its TH milieu. [source] Lysophosphatidic Acid Inhibits Ca2+ Signaling in Response to Epidermal Growth Factor Receptor Stimulation in Human Astrocytoma Cells by a Mechanism Involving Phospholipase C, and a G,i ProteinJOURNAL OF NEUROCHEMISTRY, Issue 4 2000Marita Hernández Abstract: The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca2+ concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca2+ transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase C, (PLC,)-1, and G,i protein. LPA was found to induce coupling of PLC,-1 to the EGFR by a mechanism involving a G,i protein, in the absence of tyrosine phosphorylation of both PLC, and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G,i -protein-mediated translocation of PLC,-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins. [source] Expression of hck-tr, a truncated form of the src-related tyrosine kinase hck, in bovine spermatozoa and testisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2008Louis-Jean Bordeleau Abstract In bull testicular haploid germ cells, an mRNA encoding for hck was detected in addition to another one encoding for hck-tr, a truncated form of the tyrosine kinase hck. As the transcripts were expressed in spermatids, we tried to determine whether hck-tr is present in mature bovine spermatozoa. Two polyclonal antibodies were produced against peptides specific to the N- and C-terminal portions of the truncated protein. Western blot analyses confirmed the presence of hck-tr in total protein extracts of ejaculated bull spermatozoa, and sub-cellular fractionation experiments suggest its presence in both head and flagellum. The truncated protein appears tightly associated with cytoskeletal elements as it could be extracted only with SDS under reducing conditions. When assessed by indirect immunofluorescence, hck-tr was mostly localized at the acrosomal area of the sperm cell and a similar localization was observed on demembranated spermatozoa. Immunohistochemical studies on testis sections revealed protein expression in spermatocytes as well as in round and elongating spermatids. The results presented in this study clearly show the presence of mRNAs encoding for hck and hck-tr in testicular germ cells; hck-tr being translated during spermatogenesis and expressed on mature ejaculated bull spermatozoa. Mol. Reprod. Dev. 75: 828,837, 2008. © 2007 Wiley-Liss, Inc. [source] Expression of Messenger Ribonucleic Acid Encoding for Phosphodiesterase Isoenzymes in Human Female Genital TissuesTHE JOURNAL OF SEXUAL MEDICINE, Issue 6 2007Stefan Uckert PhD ABSTRACT Objectives., The use of inhibitors of phosphodiesterase 5 (PDE5) has been suggested to treat symptoms of female sexual dysfunction (FSD). Nonetheless, there has been a relatively low success rate of PDE5 inhibitors in FSD in comparison with male erectile dysfunction. The elevated expression of PDE5 in the human penile erectile tissue is considered the reason for the high clinical efficacy of PDE5 inhibitors in the pharmacotherapy of male erectile dysfunction. Aim., To evaluate by means of molecular biology the expression of messenger ribonucleic acid expression (mRNA) encoding for cyclic AMP and cyclic GMP PDE isoenzymes in female genital tissues. Main Outcome Measures., The amount of mRNA transcripts specifically encoding for cyclic AMP- and/or cyclic GMP-degrading PDE isoenzymes was determined. Methods., Human clitoral, labial, and vaginal tissue was obtained from four female cadavers (age at death: 18,42 years). The expression of mRNA specifically encoding for PDE1A, 1B, 1C, 2A, 4A, 5A, 10A, and 11A was elucidated by means of real-time polymerase chain reaction (PCR) analysis (TaqMan). Human penile erectile tissue (corpus cavernosum [HCC]) was used as a reference tissue. Results., mRNA encoding for all PDE isoforms mentioned above is expressed in the female genital tissues. Different magnitudes of mRNA expression were observed: a predominant expression of mRNA encoding for PDE1A but only insignificant amounts of PDE1B, 1C, 4A, 10, and 11A mRNA were registered. With PDE1A being the only exception, the mRNA expression was always higher in the HCC than in the female genital tissues. Especially, the expression of mRNA encoding for PDE5 was several-fold higher in the HCC. Conclusion., On the mRNA level, various PDE isoforms are expressed in the clitoris, labia, and vagina. It remains to be established as to whether the low expression of PDE5 in female genital tissue might be a negative predictor for the success of PDE5 inhibitors in the treatment of FSD. Uckert S, Ellinghaus P, Albrecht K, Jonas U, and Oelke M. Expression of messenger ribonucleic acid encoding for phosphodiesterase isoenzymes in human female genital tissues. J Sex Med 2007;4:1604,1609. [source] Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenumBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Rutger D. Douma Abstract As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (qp) and growth rate (µ) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state qp(µ) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed-batch experiments, leads to errors in the prediction, because qp is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate-limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin-N synthase (IPNS) was the main rate-limiting enzyme for penicillin-G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady-state as well as the dynamics during chemostat start-up and fed-batch cultivation. Biotechnol. Bioeng. 2010;106: 608,618. © 2010 Wiley Periodicals, Inc. [source] Triple light chain antibodies: Factors that influence its formation in cell cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Natalia Gomez Abstract THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species,Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression,evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC),correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production,evaluated as the ratio of the cell-specific production rate of GSH (qGSH) to the cell-specific production rate of THIOMAB (qp),corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and qGSH/qp ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high qGSH/qp ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748,760. © 2009 Wiley Periodicals, Inc. [source] Non-solid oncogenes in solid tumors: EML4,ALK fusion genes in lung cancerCANCER SCIENCE, Issue 12 2008Hiroyuki Mano It is generally accepted that recurrent chromosome translocations play a major role in the molecular pathogenesis of hematological malignancies but not of solid tumors. However, chromosome translocations involving the e26 transformation-specific sequence transcription factor loci have been demonstrated recently in many prostate cancer cases. Furthermore, through a functional screening with retroviral cDNA expression libraries, we have discovered the fusion-type protein tyrosine kinase echinoderm microtubule-associated protein like-4 (EML4),anaplastic lymphoma kinase (ALK) in non-small cell lung cancer (NSCLC) specimens. A recurrent chromosome translocation, inv(2)(p21p23), in NSCLC generates fused mRNA encoding the amino-terminal half of EML4 ligated to the intracellular region of the receptor-type protein tyrosine kinase ALK. EML4,ALK oligomerizes constitutively in cells through the coiled coil domain within the EML4 region, and becomes activated to exert a marked oncogenicity both in vitro and in vivo. Break and fusion points within the EML4 locus may diverge in NSCLC cells to generate various isoforms of EML4,ALK, which may constitute ~5% of NSCLC cases, at least in the Asian ethnic group. In the present review I summarize how detection of EML4,ALK cDNA may become a sensitive diagnostic means for NSCLC cases that are positive for the fusion gene, and discuss whether suppression of ALK enzymatic activity could be an effective treatment strategy against this intractable disorder. (Cancer Sci 2008; 99: 2349,2355) [source] Transcriptional profiling of brain-derived-neurotrophic factor-induced neuronal plasticity: A novel role for nociceptin in hippocampal neurite outgrowthDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2006Robert H. Ring Abstract Brain derived neurotrophic factor (BDNF) exhibits a sequence of actions on neurons ranging from acute enhancement of transmission to long-term promotion of neurite outgrowth and synaptogenesis associated with learning and memory. The manifold effects of BDNF on neuronal modifications may be mediated by genomic alterations. We previously found that BDNF treatment acutely increases transcription of the synaptic vesicle protein Rab3A, required for trophin-induced synaptic plasticity, as well as the peptide VGF, which increases during learning. To elucidate comprehensive transcriptional programs associated with short- and long-term BDNF exposure, we now examine mRNA abundance and complexity using Affymetrix GeneChips in cultured hippocampal neurons. Consistent with the modulation of synaptic plasticity, BDNF treatment (3,6 h) induced mRNAs encoding the synapse-associated proteins synaptojanin 2, neuronal pentraxin 1, septin 9, and ryanodine receptor 2. BDNF also induced expression of mRNAs encoding neuropeptides (6,12 h), including prepronociceptin, neuropeptide Y, and secretogranin. To determine whether these neuropeptides induced by BDNF mediate neuronal development, we examined their effects on hippocampal neurons. The four mature peptides derived from post-translational processing of the ppNociceptin propeptide induced the expression of several immediate early genes in hippocampal cultures, indicating neuronal activation. To examine the significance of activation, the effects of nociceptin (orphanin FQ) and nocistatin on neurite outgrowth were examined. Quantitative morphometric analysis revealed that nociceptin significantly increased both average neurite length and average number of neurites per neuron, while nocistatin had no effect on these parameters. These results reveal a novel role for nociceptin and suggest that these neuropeptide systems may contribute to the regulation of neuronal function by BDNF. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Glutathione depletion in hippocampal cells increases levels of H and L ferritin and glutathione S-transferase mRNAsGENES TO CELLS, Issue 5 2007Nadya Morozova Glutathione plays an essential role in maintaining cellular redox balance, protecting cells from oxidative stress and detoxifying xenobiotic compounds. Glutathione depletion has been implicated in neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Cells of neuronal origin are acutely sensitive to glutathione depletion, providing an avenue for studying the mechanisms invoked for neuronal survival in response to oxidant challenge. We investigated the changes in mRNA profile in HT22 hippocampal cells following administration of homocysteic acid (HCA), a glutathione-depleting drug. We report that HCA treatment of HT22 murine hippocampal cells increases the levels of the mRNAs encoding at least three proteins involved in protection from oxidant injury, the mRNAs encoding heavy (H) and light (L) ferritin and glutathione S-transferase (GST). [source] Minocycline attenuates microglial activation but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-,JOURNAL OF NEUROCHEMISTRY, Issue 3 2006Krishnan Sriram Abstract Activated microglia are implicated in the pathogenesis of disease-, trauma- and toxicant-induced damage to the CNS, and strategies to modulate microglial activation are gaining impetus. A novel action of the tetracycline derivative minocycline is the ability to inhibit inflammation and free radical formation, factors that influence microglial activation. Minocycline is therefore being tested as a neuroprotective agent to alleviate CNS damage, although findings so far have yielded mixed results. Here, we showed that administration of a single low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (METH), a paradigm that causes selective degeneration of striatal dopaminergic nerve terminals without affecting the cell body in substantia nigra, increased the expression of mRNAs encoding microglia-associated factors F4/80, interleukin (IL)-1,, IL-6, monocyte chemoattractant protein-1 (MCP-1, CCL2) and tumor necrosis factor (TNF)-,. Minocycline treatment attenuated MPTP- or METH-mediated microglial activation, but failed to afford neuroprotection. Lack of neuroprotection was shown to be due to the inability of minocycline to abolish the induction of TNF-, and its receptors, thereby failing to modulate TNF signaling. Thus, TNF-, appeared to be an obligatory component of dopaminergic neurotoxicity. To address this possibility, we examined the effects of MPTP or METH in mice lacking genes encoding IL-6, CCL2 or TNF receptor (TNFR)1/2. Deficiency of either IL-6 or CCL2 did not alter MPTP neurotoxicity. However, deficiency of both TNFRs protected against the dopaminergic neurotoxicity of MPTP. Taken together, our findings suggest that attenuation of microglial activation is insufficient to modulate neurotoxicity as transient activation of microglia may suffice to initiate neurodegeneration. These findings support the hypothesis that TNF-, may play a role in the selective vulnerability of the nigrostriatal pathway associated with dopaminergic neurotoxicity and perhaps Parkinson's disease. [source] A novel sensor kinase,response regulator hybrid regulates type III secretion and is required for virulence in Pseudomonas aeruginosaMOLECULAR MICROBIOLOGY, Issue 4 2004Michelle A. Laskowski Summary The type III secretion system (TTSS) of Pseudomonas aeruginosa is induced by contact with eukaryotic cells and by growth in low-calcium media. We have identified a protein, RtsM, that is necessary for expression of the TTSS genes in P. aeruginosa. RtsM possesses both histidine kinase and response regulator domains common to two-component signalling proteins, as well as a large predicted periplasmic domain and seven transmembrane domains. Deletion of rtsM resulted in a defect in production and secretion of the type III effectors. Northern blot analysis revealed that mRNAs encoding the effectors ExoT and ExoU are absent in the ,rtsM strain under TTSS-inducing conditions. Using transcriptional fusions, we demonstrated that RtsM is required for transcription of the operons encoding the TTSS effectors and apparatus in response to calcium limitation or to host cell contact. The operon encoding the TTSS regulator ExsA does not respond to calcium limitation, but the basal transcription rate of this operon was lower in ,rtsM than in the wild-type parent, PA103. The defect in TTSS effector production and secretion of ,rtsM could be complemented by overexpressing ExsA or Vfr, two transcriptional activators involved in TTSS regulation. ,rtsM was markedly less virulent than PA103 in a murine model of acute pneumonia, demonstrating that RtsM is required in vivo. We propose that RtsM is a sensor protein at the start of a signalling cascade that induces expression of the TTSS in response to environmental signals. [source] Expression of hck-tr, a truncated form of the src-related tyrosine kinase hck, in bovine spermatozoa and testisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2008Louis-Jean Bordeleau Abstract In bull testicular haploid germ cells, an mRNA encoding for hck was detected in addition to another one encoding for hck-tr, a truncated form of the tyrosine kinase hck. As the transcripts were expressed in spermatids, we tried to determine whether hck-tr is present in mature bovine spermatozoa. Two polyclonal antibodies were produced against peptides specific to the N- and C-terminal portions of the truncated protein. Western blot analyses confirmed the presence of hck-tr in total protein extracts of ejaculated bull spermatozoa, and sub-cellular fractionation experiments suggest its presence in both head and flagellum. The truncated protein appears tightly associated with cytoskeletal elements as it could be extracted only with SDS under reducing conditions. When assessed by indirect immunofluorescence, hck-tr was mostly localized at the acrosomal area of the sperm cell and a similar localization was observed on demembranated spermatozoa. Immunohistochemical studies on testis sections revealed protein expression in spermatocytes as well as in round and elongating spermatids. The results presented in this study clearly show the presence of mRNAs encoding for hck and hck-tr in testicular germ cells; hck-tr being translated during spermatogenesis and expressed on mature ejaculated bull spermatozoa. Mol. Reprod. Dev. 75: 828,837, 2008. © 2007 Wiley-Liss, Inc. [source] Expression and downregulation of WNT signaling pathway genes in rhesus monkey oocytes and embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2006Ping Zheng Abstract Mammalian WNT genes encode secreted glycoproteins that are conserved homologues of the Drosophila Wingless gene, which plays a crucial role in Drosophila development. Recently, WNT pathway signaling has been implicated in ovarian development, oogenesis, and early development. We sought to evaluate whether these genes may contribute to the formation of healthy human oocytes or embryos, and whether the expression of these genes could provide informative markers of human oocyte and embryo quality. To do this, we employed the primate embryo gene expression resource (PREGER; www.preger.org) to examine expression of mRNAs encoding 38 components of the WNT signaling pathway in rhesus monkey oocytes and embryos as a nonhuman primate model. We observed considerable conservation between rhesus monkey and mouse of expression of WNT, FZD, and effector gene mRNAs, and a generalized downregulation of genes encoding key components of the WNT signaling pathway during preimplantation development. Our results support a role for WNT signaling during oocyte growth or maturation, but not during preimplantation development. Additionally, we observed differences between in vitro cultured and in vivo developing blastocysts, indicating possible effects of culture on WNT signaling during the peri-implantation period. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Trans -splicing in DrosophilaBIOESSAYS, Issue 11 2002Vincenzo Pirrotta Splicing is an efficient and precise mechanism that removes noncoding regions from a single primary RNA transcript. Cutting and rejoining of the segments occurs on nascent RNA. Trans -splicing between small specialized RNAs and a primary transcript has been known in some organisms but recent papers show that trans -splicing between two RNA molecules containing different coding regions is the normal mode in a Drosophila gene.1,3 The mod(mdg4) gene produces 26 different mRNAs encoding as many protein isoforms. The differences lie in alternative 3, exons encoded by different transcriptional units and spliced to the 5, common region by a surprising trans -splicing mechanism. BioEssays 24:988,991, 2002. © 2002 Wiley-Periodicals, Inc. [source] | |||||||||||||||||||||||||