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Protein Accumulation (protein + accumulation)
Selected AbstractsDystrophin upregulation in pressure-overloaded cardiac hypertrophy in ratsCYTOSKELETON, Issue 1 2003Masato Maeda Abstract Dystrophin is a cytoskeletal protein localized to the sarcolemma of skeletal and cardiac muscle, and neurons. We have recently demonstrated that a significant cardiac damage including myocytes injury, inflammation, and fibrosis, was found in dystrophin-deficient myocardium during pressure overload [Kamogawa et al., 2001: Cardiovasc Res 50:509,515]. However, little is known about how the cardiac sarcolemmal cytoskeleton produces qualitative and quantitative changes in response to pressure overload. Accordingly, we investigated dystrophin gene expression and protein accumulation during cardiac hypertrophy. Cardiac hypertrophy was produced by banding of the abdominal aorta of rats. Total RNA from the left ventricle of the heart was used for a quantitative reverse transcription-polymerase chain reaction (RT-PCR). Dystrophin mRNA expression significantly increased by 33 ± 18% at 1 day (P < 0.05) and 45 ± 19% at 2 days (P < 0.01) after banding, while G3PDH mRNA showed no significant change. RT-PCR for dystrophin tissue-specific exon 1 revealed that only muscle type promoter, but not non-muscle type promoter (brain and Purkinje-cell type), was activated immediately after banding. Immunohistochemistry for dystrophin showed intense cellular membrane staining with an increase in the perimeter of the myocytes by 14% at 3 days (46.3 ,m, P < 0.01) and 19% at 7 days (51.2 ,m, P < 0.01) after banding. Western blotting also showed dystrophin protein increased by 14 ± 6% at 2 days (P < 0.05) and by 32 ± 10% at 3 days (P < 0.01) after aortic banding. In conclusion, upregulation of dystrophin mRNA expression and protein accumulation occurs in response to cardiac hypertrophy. These data and the vulnerability of dystrophin-deficient myocardium to pressure overload suggest that dystrophin could play an important role in maintaining the integrity of the sarcolemma. Cell Motil. Cytoskeleton 55:26,35, 2003. © 2003 Wiley-Liss, Inc. [source] Involvement of hypoxia-inducible factor-1 HiF(1,) in IgE-mediated primary human basophil responsesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2009Vadim V. Sumbayev Abstract Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE-mediated activation of primary human basophils results in protein accumulation of the ,-subunit of hypoxia-inducible factor 1, (HIF-1,), which is differentially regulated compared with signals controlling histamine release. HIF-1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3-kinase, were critical for IgE-mediated accumulation of HIF-1,, although the latter crucially affected degranulation. Abrogating HIF-1, expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF-1, protein alters IgE-induced ATP depletion in basophils, thus also supporting the production of the pro-allergic cytokine IL-4. [source] Pertussis toxin activates adult and neonatal naive human CD4+ T,lymphocytesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2006Sandrine Tonon Abstract Pertussis toxin (PTX) is known to be mitogenic for T,lymphocytes, but its direct action on naive human T cells has not been specified. Herein, we show that PTX induces the proliferation of purified adult CD45RA+CD4+ T cells independently of its ADP-ribosyltransferase activity. PTX directly induces TNF-, and IL-2 mRNA expression, modulates the level of several cell surface receptors and induces Forkhead box,p3 (Foxp3) protein accumulation in naive CD4+ T cells. Addition of autologous dendritic cells was found to be required for the production of high levels of IFN-, by PTX-stimulated naive T cells. These effects of PTX occurred in conjunction with activation of NF-,B and NFAT transcription factors. Overall, responses of neonatal CD4+ T cells to PTX were similar to those of adult CD45RA+CD4+ naive T cells except for their blunted CD40 ligand up-regulation. We suggest that the adjuvant properties of PTX during primary cell-mediated immune responses involve a direct action on naive T,lymphocytes in addition to activation of antigen-presenting cells. [source] An evolutionary transition of vasa regulation in echinodermsEVOLUTION AND DEVELOPMENT, Issue 5 2009Celina E. Juliano SUMMARY Vasa, a DEAD box helicase, is a germline marker that may also function in multipotent cells. In the embryo of the sea urchin Strongylocentrotus purpuratus, Vasa protein is posttranscriptionally enriched in the small micromere lineage, which results from two asymmetric cleavage divisions early in development. The cells of this lineage are subsequently set aside during embryogenesis for use in constructing the adult rudiment. Although this mode of indirect development is prevalent among echinoderms, early asymmetric cleavage divisions are a derived feature in this phylum. The goal of this study is to explore how vasa is regulated in key members of the phylum with respect to the evolution of the micromere and small micromere lineages. We find that although striking similarities exist between the vasa mRNA expression patterns of several sea urchins and sea stars, the time frame of enriched protein expression differs significantly. These results suggest that a conserved mechanism of vasa regulation was shifted earlier in sea urchin embryogenesis with the derivation of micromeres. These data also shed light on the phenotype of a sea urchin embryo upon removal of the Vasa-positive micromeres, which appears to revert to a basal mechanism used by extant sea stars and pencil urchins to regulate Vasa protein accumulation. Furthermore, in all echinoderms tested here, Vasa protein and/or message is enriched in the larval coelomic pouches, the site of adult rudiment formation, thus suggesting a conserved role for vasa in undifferentiated multipotent cells set aside during embryogenesis for use in juvenile development. [source] Increased temperature and protein oxidation lead to HSP72 mRNA and protein accumulation in the in vivo exercised rat heartEXPERIMENTAL PHYSIOLOGY, Issue 1 2009Jessica L. Staib Expression of myocardial heat shock protein 72 (HSP72), mediated by its transcription factor, heat shock factor 1 (HSF1), increases following exercise. However, the upstream stimuli governing exercise-induced HSF1 activation and subsequent Hsp72 gene expression in the whole animal remain unclear. Exercise-induced increases in body temperature may promote myocardial radical production, leading to protein oxidation. Conceivably, myocardial protein oxidation during exercise may serve as an important signal to promote nuclear HSF1 migration and activation of Hsp72 expression. Therefore, these experiments tested the hypothesis that prevention of exercise-induced increases in body temperature attenuates cardiac protein oxidation, diminishes HSF1 activation and decreases HSP72 expression in vivo. To test this hypothesis, in vivo exercise-induced changes in body temperature were manipulated by exercising male rats in either cold (4°C) or warm ambient conditions (22°C). Warm exercise increased both body temperature (+3°C) and myocardial protein oxidation, whereas these changes were attenuated by cold exercise. Interestingly, exercise in both conditions did not significantly increase myocardial nuclear localized phosphorylated HSF1. Nonetheless, warm exercise elevated left-ventricular HSP72 mRNA by ninefold and increased myocardial HSP72 protein levels by threefold compared with cold-exercised animals. Collectively, these data indicate that elevated body temperature and myocardial protein oxidation promoted exercise-induced cardiac HSP72 mRNA expression and protein accumulation following in vivo exercise. However, these results suggest that exercise-induced myocardial HSP72 protein accumulation is not a result of nuclear-localized, phosphorylated HSF1, indicating that other transcriptional or post-transcriptional regulatory mechanisms are involved in exercise-induced HSP72 expression. [source] [Na+]i -induced c-Fos expression is not mediated by activation of the 5,-promoter containing known transcriptional elementsFEBS JOURNAL, Issue 14 2007Mounsif Haloui In vascular smooth muscle cells and several other cell types, inhibition of Na+/K+ -ATPase leads to the expression of early response genes, including c-Fos. We designed this study to examine whether or not a putative Na+i/K+i -sensitive element is located within the c-Fos 5,-UTR from ,,650 to +,103 containing all known response elements activated by ,classic' stimuli, such as growth factors and Ca2+i -raising compounds. In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na+/K+ -ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na+i and loss of K+i. Augmented c-Fos expression was also observed under inhibition of Na+/K+ -ATPase in K+ -free medium and in the presence of the Na+ ionophore monensin. The effect of ouabain on c-Fos expression was sharply attenuated under dissipation of the transmembrane Na+ gradient, but was preserved in the presence of Ca2+ chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na+i -mediated, Ca2+i - and extracellular regulated kinase-independent mechanism of gene expression. In contrast to massive c-Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c-Fos 5,-UTR. Negative results were also obtained in ouabain-treated vascular smooth muscle cells and C11 Madin,Darby canine kidney cells possessing augmented c-Fos expression. Our results reveal that Na+i -induced c-Fos expression is not mediated by the 5,-UTR containing transcriptional elements activated by growth factors and other ,classic stimuli'. [source] Quantitative isolation of ,1AT mutant Z protein polymers from human and mouse livers and the effect of heat,HEPATOLOGY, Issue 1 2005Jae-Koo An Alpha-1-antitrypsin (,1AT) deficiency in its most common form is caused by homozygosity for the ,1AT mutant Z gene. This gene encodes a mutant Z secretory protein, primarily synthesized in the liver, that assumes an abnormal conformation and accumulates within hepatocytes causing liver cell injury. Studies have shown that mutant ,1ATZ protein molecules form unique protein polymers. These Z protein polymers have been hypothesized to play a critical role in the pathophysiology of liver injury in this disease, although a lack of quantitative methods to isolate the polymers from whole liver has hampered further analysis. In this study, we demonstrate a quantitative ,1ATZ polymer isolation technique from whole liver and show that the hepatocellular periodic acid-Schiff,positive globular inclusions that are the histopathological hallmark of this disease are composed almost entirely of the polymerized ,1ATZ protein. Furthermore, we examine the previously proposed but untested hypothesis that induction of ,1ATZ polymerization by the heat of physiological fever is part of the mechanism of hepatic ,1ATZ protein accumulation. The results, however, show that fever-range temperature elevations have no detectable effect on steady-state levels of intrahepatic Z protein polymer in a model in vivo system. In conclusion, methods to separate insoluble protein aggregates from liver can be used for quantitative isolation of ,1ATZ protein polymers, and the effect of heat from physiological fever may be different in vivo compared with in vitro systems. (HEPATOLOGY 2005;41:160,167.) [source] Repeated grazing of a salt marsh grass by moulting greylag geese Anser anser, does sequential harvesting optimise biomass or protein gain?JOURNAL OF AVIAN BIOLOGY, Issue 1 2003Anthony D. Fox The effects of simulated goose grazing on common saltmarsh-grass Puccinellia maritima plants were tested on a Danish salt marsh during the flightless moulting period of greylag geese Anser anser (3,21 June 1998). Plants in an area exclosed from the influence of grazing and the nutrient effects of goose faeces were subject to removal of youngest lamina at 3-, 6-, 9- and 18-day intervals during this period. Average biomass and protein accumulation between harvests was highest at defoliation intervals of 9 days or more. Field observations from two separate study areas demonstrated geese returned to regraze the Puccinellia sward after 6,8 days and oesophageal contents from feeding geese showed selection for lamina lengths consistent with the results of clipping every 6 days. Geese therefore regrazed Puccinellia patches at shorter intervals than expected were they to maximise their intake of biomass or protein at each visit. However, total cumulative lamina elongation, equivalent to the long term gain during the entire moult period, showed no significant difference between the three most intensive defoliation treatments, which were significantly greater than those of plants defoliated at 18 day intervals. Highest overall lamina protein levels were maintained at 6- and 9-day defoliation intervals. This suggests geese regrazed Puccinellia patches at a rate that maximised their number of harvests during the flightless period, but maintained highest protein levels and overall biomass in the sward. This suggests, in line with earlier studies, that moulting greylag geese combine dietary selection, reduced nitrogen excretion and regrazing patterns to meet protein demands during regrowth of flight feathers. [source] Dysfunction of the unfolded protein response increases neurodegeneration in aged rat hippocampus following proteasome inhibitionAGING CELL, Issue 6 2009María Paz Gavilán Summary Dysfunctions of the ubiquitin proteasome system (UPS) have been proposed to be involved in the aetiology and/or progression of several age-related neurodegenerative disorders. However, the mechanisms linking proteasome dysfunction to cell degeneration are poorly understood. We examined in young and aged rat hippocampus the activation of the unfolded protein response (UPR) under cellular stress induced by proteasome inhibition. Lactacystin injection blocked proteasome activity in young and aged animals in a similar extent and increased the amount of ubiquitinated proteins. Young animals activated the three UPR arms, IRE1,, ATF6, and PERK, whereas aged rats failed to induce the IRE1, and ATF6, pathways. In consequence, aged animals did not induce the expression of pro-survival factors (chaperones, Bcl-XL and Bcl-2), displayed a more sustained expression of pro-apoptotic markers (CHOP, Bax, Bak and JKN), an increased caspase-3 processing. At the cellular level, proteasome inhibition induced neuronal damage in young and aged animals as assayed using Fluorojade-B staining. However, degenerating neurons were evident as soon as 24 h postinjection in aged rats, but it was delayed up to 3 days in young animals. Our findings show evidence supporting age-related dysfunctions in the UPR activation as a potential mechanism linking protein accumulation to cell degeneration. An imbalance between pro-survival and pro-apoptotic proteins, because of noncanonical activation of the UPR in aged rats, would increase the susceptibility to cell degeneration. These findings add a new molecular vision that might be relevant in the aetiology of several age-related neurodegenerative disorders. [source] FOURIER TRANSFORM INFRARED SPECTROSCOPY AS A NOVEL TOOL TO INVESTIGATE CHANGES IN INTRACELLULAR MACROMOLECULAR POOLS IN THE MARINE MICROALGA CHAETOCEROS MUELLERII (BACILLARIOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 2 2001Mario Giordano Fourier Transform Infrared (FT-IR) spectroscopy was used to study carbon allocation patterns in response to changes in nitrogen availability in the diatom Chaetoceros muellerii Lemmerman. The results of the FT-IR measurements were compared with those obtained with traditional chemical methods. The data obtained with both FT-IR and chemical methods showed that nitrogen starvation led to the disappearance of the differences in cell constituents and growth rates existing between cells cultured at either high [NO3,] or high [NH4+]. Irrespective of the nitrogen source supplied before nitrogen starvation, a diversion of carbon away from protein, chlorophyll, and carbohydrates into lipids was observed. Under these conditions, cells that had previously received nitrogen as nitrate appeared to allocate a larger amount of mobilized carbon into lipids than cells that had been cultured in the presence of ammonia. All these changes were reversed by resupplying the cultures with nitrogen. The rate of protein accumulation in the N-replete cells was slower than the rate of decrease under nitrogen starvation. This study demonstrates that the relative proportions of the major macromolecules contained in microalgal cells and their changes in response to external stimuli can be determined rapidly, simultaneously, and inexpensively using FT-IR. The technique proved to be equally reliable to and less labor intensive than more traditional chemical methods. [source] Programmed responses to virus replication in plantsMOLECULAR PLANT PATHOLOGY, Issue 1 2000A. J. Maule Despite their economic importance, we understand very little about the mechanism leading to symptom formation in compatible virus infections. By applying a spatial analysis to advancing infection fronts, we have been able to relate molecular events in small groups of cells to a sequence of virus-induced changes. This sequence starts ahead of the main front of virus replication and virus protein accumulation and lasts beyond the time at which virus replication has ceased. The host changes include alterations in gene expression, physiology and cellular ultrastructure. The relationship between these effects has been analysed in comparative studies between different virus infections in different hosts and abiotic stress. The research points to there being common features for different viruses leading to common effects. Also, although many of the consequences of virus infection are similar to the effects of heat shock, there are sufficient differences to suggest that the two inducers use distinct control pathways. The immediate challenge for the future is to establish synchronous infections of tissues so that the complex relationship between the virus and the host can be investigated using temporal rather than spatial analyses. [source] Squamous cell carcinoma arising from a seminal vesicular cyst: Possible relationship between chronic inflammation and tumor developmentPATHOLOGY INTERNATIONAL, Issue 3 2002Nobuyuki Yanagisawa A case of squamous cell carcinoma arising within an acquired seminal vesicular cyst is described. A 61-year-old man was hospitalized because of hemospermia and dysuria. Under the diagnosis of a left seminal vesicular cyst, surgical resection was performed. Pathological examination revealed squamous cell carcinoma within a seminal vesicular cyst, along with squamous metaplastic foci and severe chronic inflammation. Cell proliferation, determined with reference to MIB-1 labeling indices, showed a stepwise increase from normal columnar epithelium, through squamous metaplasia, to squamous cell carcinoma. Sporadic p53 protein accumulation without evident gene mutations was also apparent in both the carcinoma and squamous metaplastic lesions. We therefore concluded that the squamous cell carcinoma might have developed from squamous metaplastic foci associated with chronic inflammatory stimulation, within a seminal vesicular cyst. [source] Sunscreens containing the broad-spectrum UVA absorber, Mexoryl® SX, prevent the cutaneous detrimental effects of UV exposure: a review of clinical study resultsPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 4 2008Anny Fourtanier Background: UVA exposure of human skin mainly produces reactive oxygen species (ROS) leading to DNA, cell and tissue damage. It alters immune function, pigmentation and it is certainly responsible for a large part of photoaging changes. Moreover UVA is implicated in the etiology of several photodermatoses. As a consequence, to provide adequate protection, sunscreens or skin care products for daily use protective products need UVA absorbers combined with UVB ones. Aim: To assess the efficacy of sunscreens containing a broad-spectrum UVA absorber the Mexoryl® SX or ecamsule and to compare formulations with and without it through a large number of clinical studies in human volunteers and patients. Methods: The following assessments were conducted: ,Prevention of excessive pigmentation induced by UV exposure in Caucasian and Asian skins using a method that measures pigmentation protection factors (PPF). ,Efficacy against DNA damage by measurement of pyrimidine dimer formation and p53 protein accumulation. ,Protection of immune system using delayed type hypersensitivity (DTH) reactions to recall antigens, isomerization of urocanic acid (UCA), alteration of Langerhans cells (LC) density, morphology and function. ,Reduction of epidermal and dermal alterations induced by repeated UVA or UV solar simulated radiation (SSR) using histology or immunohistology. ,Prevention of the polymorphous light eruption (PMLE) in patients prone to develop this disease. Results: Mexoryl® SX-containing formulations showed a dose-dependent level of protection against pigmentation. For a same sun protection factor (SPF) the higher the UVA protection was, the higher was the PPF. Pyrimidine dimer formation and p53 accumulation were significantly reduced by formulations with Mexoryl® SX. In the studies looking at the suppression of DTH reactions to recall antigens by the different UV spectra, the LC alterations and the cis UCA formation, Mexoryl® SX formulations always showed a higher protective potency than sunscreen without it even when the protection against erythema was similar (products with same SPF). Mexoryl® SX formulations also prevented or significantly decreased to minimal, ferritin, tenascin and lysozyme expression induced by repeated UVA or SSR exposure. It also reduced the enhancement of collagenase 2 mRNA expression induced by SSR exposure. Finally PMLE study demonstrated that UVA protection was essential for the prevention of this photodermatose. Conclusion: Mexoryl® SX formulated in sunscreens or daily use products have been shown to be an effective UV absorber, leading to an increased efficacy of these products against a large number of biological damage induced by UVA, SSR or sun exposure. [source] Accumulated p53 protein and UVA protection level of sunscreensPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 1 2000S. Seité Nuclear p53 expression is a sensitive parameter for the detection of ultraviolet (UV)-induced skin damage, and it has been used as an endpoint to evaluate the effectiveness of sunscreens. In this study, we compared the protection provided by two sunscreens having identical sun protection factors (SPF) but different UVA protection factors (UVA-PF) measured by the persistent pigment darkening method (PPD). The SPF of the sunscreens was 7 and the UVA-PF were respectively 7 and 3. Nuclear p53 protein was quantified in human skin biopsies treated with sunscreens and exposed 8 times to 5 MED of solar simulated radiation (SSR). The results showed that both sunscreens offered only partial protection against the increased expression of nuclear p53 protein induced by repetitive SSR exposures. However, a significantly lower level of p53-positive cells was found in areas protected with the sunscreen having the higher UVA-PF compared to the other sunscreen protected areas. In order to verify whether the difference in efficacy of these products was due to the difference in UVA absorption capacity, we quantified epidermal p53 protein accumulation after 8 exposures to either UVA (320,400 nm) or UVA1 (340,400 nm). We showed that as with SSR, repetitive exposures to 12.5 and 25 J/cm2 of UVA or UVA1 induced a significant increase in p53-positive cells in the human epidermis. These results confirmed that SPF determined on the basis of an acute erythemal reaction does not predict the level of protection against cumulative damage. They also showed that the protection provided by two sunscreens with different UVA protection factors is different (based on nuclear p53 protein accumulation), and that the PPD method can distinguish varying levels of sunscreen efficacy against UVA-induced cell damage. [source] Papaya fruit softening, endoxylanase gene expression, protein and activityPHYSIOLOGIA PLANTARUM, Issue 3 2007Ashariya Manenoi Papaya (Carica papaya L.) cell wall matrix polysaccharides are modified as the fruit starts to soften during ripening and an endoxylanase is expressed that may play a role in the softening process. Endoxylanase gene expression, protein amount and activity were determined in papaya cultivars that differ in softening pattern and in one cultivar where softening was modified by the ethylene receptor inhibitor 1-methylcyclopropene (1-MCP). Antibodies to the endoxylanase catalytic domain were used to determine protein accumulation. The three papaya varieties used in the study, ,Line 8', ,Sunset', and ,Line 4-16', differed in softening pattern, respiration rate, ethylene production and showed similar parallel relationships during ripening and softening in endoxylanase expression, protein level and activity. When fruit of the three papaya varieties showed the respiratory climacteric and started to soften, the level of endoxylanase gene expression increased and this increase was related to the amount of endoxylanase protein at 32 kDa and its activity. Fruit when treated at less than 10% skin yellow stage with 1-MCP showed a significant delay in the respiratory climacteric and softening, and reduced ethylene production, and when ripe was firmer and had a ,rubbery' texture. The 1-MCP-treated fruit that had the ,rubbery' texture showed suppressed endoxylanase gene expression, protein and enzymatic activity. Little or no delay occurred between endoxylanase gene expression and the appearance of activity during posttranslational processing from 65 to 32 kDa. The close relationship between endoxylanase gene expression, protein accumulation and activity in different varieties and the failure of the 1-MCP-treated fruit to fully soften, supported de novo synthesis of endoxylanase, rapid posttranslation processing and a role in papaya fruit softening. [source] Approaches to achieve high-level heterologous protein production in plantsPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2007Stephen J. Streatfield Summary Plants offer an alternative to microbial fermentation and animal cell cultures for the production of recombinant proteins. For protein pharmaceuticals, plant systems are inherently safer than native and even recombinant animal sources. In addition, post-translational modifications, such as glycosylation, which cannot be achieved with bacterial fermentation, can be accomplished using plants. The main advantage foreseen for plant systems is reduced production costs. Plants should have a particular advantage for proteins produced in bulk, such as industrial enzymes, for which product pricing is low. In addition, edible plant tissues are well suited to the expression of vaccine antigens and pharmaceuticals for oral delivery. Three approaches have been followed to express recombinant proteins in plants: expression from the plant nuclear genome; expression from the plastid genome; and expression from plant tissues carrying recombinant plant viral sequences. The most important factor in moving plant-produced heterologous proteins from developmental research to commercial products is to ensure competitive production costs, and the best way to achieve this is to boost expression. Thus, considerable research effort has been made to increase the amount of recombinant protein produced in plants. This research includes molecular technologies to increase replication, to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations optimal for their accumulation, and to engineer proteins to stabilize them. Other methods include plant breeding to increase transgene copy number and to utilize germplasm suited to protein accumulation. Large-scale commercialization of plant-produced recombinant proteins will require a combination of these technologies. [source] Identification of a 150 bp cis -acting element of the AtNRT2.1 promoter involved in the regulation of gene expression by the N and C status of the plantPLANT CELL & ENVIRONMENT, Issue 11 2007THOMAS GIRIN ABSTRACT The Arabidopsis thaliana AtNRT2.1 gene, which encodes a NO3 - transporter involved in high-affinity uptake by the roots, is a molecular target of several mechanisms responsible for the regulation of root NO3 - acquisition by the N status of the plant. All levels of AtNRT2.1 expression (promoter activity, transcript level, protein accumulation, transport activity) are coordinately up-regulated in the presence of NO3 - , and repressed by downstream N metabolites. Transgenic plants expressing the GUS reporter gene under the control of upstream sequences of AtNRT2.1 have been studied to identify elements targeted by these two regulatory mechanisms. A 150 bp sequence located upstream of the TATA box that is required for both stimulation by NO3 - and repression by N metabolites of the promoter has been identified. This sequence is able to confer these two regulations to a minimal promoter. Split-root experiments indicate that the stimulation of the chimaeric promoter by NO3 - occurs only at the local level, whereas its repression by N metabolites is mediated by a systemic signal spread to the whole plant. The activity of the cis -acting 150 bp element is also regulated by sucrose supply to the roots, suggesting a possible interaction between N and C signalling within this short region. Accordingly, multiple motifs potentially involved in regulations by N and/or C status are identified within this sequence by bioinformatic approaches. This is the first report of such a cis -acting element in higher plants. [source] Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf bladesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2006Tariq Mahmood Abstract Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonasoryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight. [source] The chloroplastic lipocalin AtCHL prevents lipid peroxidation and protects Arabidopsis against oxidative stressTHE PLANT JOURNAL, Issue 4 2009Gabriel Levesque-Tremblay Summary Lipocalins are small ligand-binding proteins with a simple tertiary structure that gives them the ability to bind small, generally hydrophobic, molecules. Recent studies have shown that animal lipocalins play important roles in the regulation of developmental processes and are involved in tolerance to oxidative stress. Plants also possess various types of lipocalins, and bioinformatics analyses have predicted that some lipocalin members may be present in the chloroplast. Here we report the functional characterization of the Arabidopsis thaliana chloroplastic lipocalin AtCHL. Cellular fractionation showed that AtCHL is a thylakoid lumenal protein. Drought, high light, paraquat and abscisic acid treatments induce AtCHL transcript and protein accumulation. Under normal growth conditions, knockout (KO) and over-expressing (OEX) lines do not differ from wild-type plants in terms of phenotype and photosynthetic performance. However, KO plants, which do not accumulate AtCHL, show more damage upon photo-oxidative stress induced by drought, high light or paraquat. In contrast, a high level of AtCHL allows OEX plants to cope better with these stress conditions. When exposed to excess light, KO plants display a rapid accumulation of hydroxy fatty acids relative to the wild-type, whereas the lipid peroxidation level remains very low in OEX plants. The increased lipid peroxidation in KO plants is mediated by singlet oxygen and is not correlated with photo-inhibition of the photosystems. This work provides evidence suggesting that AtCHL is involved in the protection of thylakoidal membrane lipids against reactive oxygen species, especially singlet oxygen, produced in excess light. [source] Repression of light signaling by Arabidopsis SPA1 involves post-translational regulation of HFR1 protein accumulationTHE PLANT JOURNAL, Issue 1 2005Jianping Yang Summary Arabidopsis uses two major classes of photoreceptors to mediate seedling de-etiolation. The cryptochromes (cry1 and cry2) absorb blue/ultraviolet-A light, whereas the phytochromes (phyA,phyE) predominantly regulate responses to red/far-red light. Arabidopsis COP1 represses light signaling by acting as an E3 ubiquitin ligase in the nucleus, and is responsible for targeted degradation of a number of photomorphogenesis-promoting factors, including HY5, LAF1, phyA, and HFR1. Distinct light signaling pathways initiated by multiple photoreceptors (including both phytochromes and cryptochromes) eventually converge on COP1, causing its inactivation and nuclear depletion. Arabidopsis SPA1, which encodes a protein structurally related to COP1, also represses light signaling under various light conditions. In this study, we present genetic evidence supporting that HFR1, which encodes a photomorphogenesis-promoting bHLH transcription factor, acts downstream of SPA1 and is required for different subsets of branch pathways of light signaling controlled by SPA1 under different light conditions. We show that SPA1 physically interacts with HFR1 in a yeast two-hybrid assay and an in vitro co-immunoprecipitation assay. We demonstrate that higher levels of HFR1 protein accumulate in the spa1 mutant background under various light conditions, including far-red, red, blue, and white light, whereas a marginal increase in HFR1 transcript level is only seen in dark- and far-red light-grown spa1-100 mutants. Together, our data suggest that repression of light signaling by Arabidopsis SPA1 likely involves post-translational regulation of HFR1 protein accumulation. [source] Early frontotemporal dementia targets neurons unique to apes and humansANNALS OF NEUROLOGY, Issue 6 2006William W. Seeley MD Objective Frontotemporal dementia (FTD) is a neurodegenerative disease that erodes uniquely human aspects of social behavior and emotion. The illness features a characteristic pattern of early injury to anterior cingulate and frontoinsular cortex. These regions, though often considered ancient in phylogeny, are the exclusive homes to the von Economo neuron (VEN), a large bipolar projection neuron found only in great apes and humans. Despite progress toward understanding the genetic and molecular bases of FTD, no class of selectively vulnerable neurons has been identified. Methods Using unbiased stereology, we quantified anterior cingulate VENs and neighboring Layer 5 neurons in FTD (n = 7), Alzheimer's disease (n = 5), and age-matched nonneurological control subjects (n = 7). Neuronal morphology and immunohistochemical staining patterns provided further information about VEN susceptibility. Results FTD was associated with early, severe, and selective VEN losses, including a 74% reduction in VENs per section compared with control subjects. VEN dropout was not attributable to general neuronal loss and was seen across FTD pathological subtypes. Surviving VENs were often dysmorphic, with pathological tau protein accumulation in Pick's disease. In contrast, patients with Alzheimer's disease showed normal VEN counts and morphology despite extensive local neurofibrillary pathology. Interpretation VEN loss links FTD to its signature regional pattern. The findings suggest a new framework for understanding how evolution may have rendered the human brain vulnerable to specific forms of degenerative illness. Ann Neurol 2006;60:660,667 [source] Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free systemBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Marcus E. Boyer Abstract The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37°C, Fd accumulated to >450 µg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28°C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system. © 2006 Wiley Periodicals, Inc. [source] Involvement of Clusterin and the Aggresome in Abnormal Protein Deposits in Myofibrillar Myopathies and Inclusion Body MyositisBRAIN PATHOLOGY, Issue 2 2005I. Ferrer Myofibrillar myopathies (MM) are characterized morphologically by the presence of non-hyaline structures corresponding to foci of dissolution of myofibrils, and hyaline lesions composed of aggregates of compacted and degraded myofibrillar elements. Inclusion body myositis (IBM) is characterized by the presence of rimmed vacuoles, eosinophilic inclusions in the cytoplasm, rare intranuclear inclusions, and by the accumulation of several abnormal proteins. Recent studies have demonstrated impaired proteasomal expression and activity in MM and IBM, thus accounting, in part, for the abnormal protein accumulation in these diseases. The present study examines other factors involved in protein aggregation in MM and IBM. Clusterin is a multiple-function protein which participates in A,-amyloid, PrPres and ,-synuclein aggregation in Alzheimer disease, prionopathies and ,-synucleinopathies, respectively. ,-Tubulin is present in the centrosome and is an intracellular marker of the aggresome. Moderate or strong clusterin immunoreactivity has been found in association with abnormal protein deposits, as revealed by immunohistochemistry, single and double-labeling immunofluorescence and confocal microscopy, in MM and IBM, and in target structures in denervation atrophy. ,-Tubulin has also been observed in association with abnormal protein deposits in MM, IBM, and in target fibers in denervation atrophy. These morphological findings are accompanied by increased expression of clusterin and ,-tubulin in muscle homogenates of MM and IBM cases, as revealed by gel electrophoresis and Western blots. Together, these observations demonstrate involvement of clusterin in protein aggregates, and increased expression of aggresome markers in association with abnormal protein inclusions in MM and IBM and in targets, as crucial events related with the pathogenesis of abnormal protein accumulation and degradation in these muscular diseases. [source] Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001Aikaterini Kontrogianni-Konstantopoulos Abstract SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (,P) and the other a truncation of the C-terminal F-domain (,F), revealed that ,P is localized similarly to full-length receptor, whereas ,F is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas ,P does not bind DNA and ,F binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development. Mol. Reprod. Dev. 60: 147,157, 2001. © 2001 Wiley-Liss, Inc. [source] Brainstem pathology in spasmodic dysphonia,THE LARYNGOSCOPE, Issue 1 2010Kristina Simonyan MD Abstract Spasmodic dysphonia (SD) is a primary focal dystonia of unknown pathophysiology, characterized by involuntary spasms in the laryngeal muscles during speech production. We examined two rare cases of postmortem brainstem tissue from SD patients compared to four controls. In the SD patients, small clusters of inflammation were found in the reticular formation surrounding solitary tract, spinal trigeminal, and ambigual nuclei, inferior olive, and pyramids. Mild neuronal degeneration and depigmentation were observed in the substantia nigra and locus coeruleus. No abnormal protein accumulations and no demyelination or axonal degeneration were found. These neuropathological findings may provide insights into the pathophysiology of SD. Laryngoscope, 2010 [source] The nature of tobacco resistance against Botrytis cinerea depends on the infection structures of the pathogenENVIRONMENTAL MICROBIOLOGY, Issue 1 2010Mohamed El Oirdi Summary To protect themselves, plants have evolved an armoury of defences in response to pathogens and other stress situations. These include the production of pathogenesis-related (PR) proteins and the accumulation of antimicrobial molecules such as phytoalexins. Here we report that resistance of tobacco to Botrytis cinerea is cultivar specific. Nicotiana tabacum cv. Petit Havana but not N. tabacum cv. Xanthi or cv. samsun is resistant to B. cinerea. This resistance is correlated with the accumulation of the phytoalexin scopoletin and PR proteins. We also show that this resistance depends on the type of B. cinerea stage. Nicotiana tabacum cv. Petit Havana is more resistant to spores than to mycelium of B. cinerea. This reduced resistance of N. tabacum cv. Petit Havana to the mycelium compared with spores is correlated with the suppression of PR proteins accumulation and the capacity of the mycelium, not the spores, to metabolize scopoletin. These data present an important advance in understanding the strategies used by B. cinerea to establish its disease on tobacco plants. [source] Temperature- and pH-dependent accumulation of heat-shock proteins in the acidophilic green alga Chlamydomonas acidophilaFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Antje Gerloff-Elias Abstract Chlamydomonas acidophila, a unicellular green alga, is a dominant phytoplankton species in acidic water bodies, facing severe environmental conditions such as low pH and high heavy metal concentrations. We examined the pH-, and temperature-dependent accumulation of heat-shock proteins in this alga to determine whether heat-shock proteins play a role in adaptation to their environment. Our results show increased heat-shock proteins accumulation at suboptimal pHs, which were not connected with any change in intracellular pH. In comparison to the mesophilic Chlamydomonas reinhardtii, the acidophilic species exhibited significantly higher accumulations of heat-shock proteins under control conditions, indicating an environmental adaptation of increased basal levels of heat-shock proteins. The results suggest that heat-shock proteins might play a role in the adaptation of C. acidophila, and possibly other acidophilic algae, to their extreme environment. [source] | ||||||||||||||||||||||||||||