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Inactivation
Kinds of Inactivation Terms modified by Inactivation Selected AbstractsSEXUAL ANTAGONISM AND THE EVOLUTION OF X CHROMOSOME INACTIVATIONEVOLUTION, Issue 8 2008Jan Engelstädter In most female mammals, one of the two X chromosomes is inactivated early in embryogenesis. Expression of most genes on this chromosome is shut down, and the inactive state is maintained throughout life in all somatic cells. It is generally believed that X-inactivation evolved as a means of achieving equal gene expression in males and females (dosage compensation). Following degeneration of genes on the Y chromosome, gene expression on X chromosomes in males and females is upregulated. This results in closer to optimal gene expression in males, but deleterious overexpression in females. In response, selection is proposed to favor inactivation of one of the X chromosomes in females, restoring optimal gene expression. Here, we make a first attempt at shedding light on this intricate process from a population genetic perspective, elucidating the sexually antagonistic selective forces involved. We derive conditions for the process to work and analyze evolutionary stability of the system. The implications of our results are discussed in the light of empirical findings and a recently proposed alternative hypothesis for the evolution of X-inactivation. [source] SELF-IMPOSED SILENCE: PARENTAL ANTAGONISM AND THE EVOLUTION OF X-CHROMOSOME INACTIVATIONEVOLUTION, Issue 3 2006David Haig Abstract A model is proposed for the evolution of X-chromosome inactivation (XCI) in which natural selection initially favors the silencing of paternally derived alleles of X-linked demand inhibitors. The compensatory upregulation of maternally derived alleles establishes a requirement for monoallelic expression in females. For this reason, XCI is self-reinforcing once established. However, inactivation of a particular X chromosome is not. Random XCI (rXCI) is favored over paternal XCI because rXCI reduces the costs of functional hemizygosity in females. Once present, rXCI favors the evolution of locus-by-locus imprinting of X-linked loci, which creates an evolutionary dynamic in which different chromosomes compete to remain active. [source] HIGH PRESSURE INACTIVATION OF PECTIN METHYL ESTERASE IN ORANGE JUICE USING COMBINATION TREATMENTSJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2001S. BASAK ABSTRACT The contribution of several high pressure (HP) processing related factors (pressure level, 300-400 MPa; pressure cycle, 1-3, and pressure-hold time, 30,120 min) on the inactivation of pectin methyl esterase (PME) in single strength (pH 3.7 and 11.4 °Brix) and concentrated (pH 3.5 and 42 °Brix) orange juice was evaluated. A response surface methodology was employed to model the combined effects of factors on the enzyme inactivation. The main effects were described by linear or quadratic functions. For both single strength and concentrated orange juices, the effects of all three main factors and some interactions (pressure level, cycle and holding time) were statistically significant (p<0.05). The dual nature of pressure inactivation of PME (with an instantaneous inactivation due to a pressure pulse, instantaneous pressure fall, and first order rate of inactivation during the pressure hold, yielding D and z values) reported in earlier studies was confirmed. Combination models were developed to predict the residual enzyme activity as influenced by the pressure level, number of pressure cycles and pressure hold time. [source] INACTIVATION OF STAPHYLOCOCCUS AUREUS EXPOSED TO DENSE-PHASE CARBON DIOXIDE IN A BATCH SYSTEMJOURNAL OF FOOD PROCESS ENGINEERING, Issue 1 2009HUACHUN HUANG ABSTRACT The inactivation of Staphylococcus aureus exposed to dense-phase carbon dioxide (DPCD) was investigated, and the kinetics of come-up time (CUT) in pressurization was monitored with come-down time (CDT) and temperature fluctuation in depressurization. CUT was about 2.5, 3.5, 4.0 and 4.0 min; CDT was 3.4, 3.7, 4.5 and 4.5 min; lowest temperature of samples in depressurization was 4, ,1, ,15 and ,22C, corresponding to 10, 20, 30 and 40 MPa at 37C. The inactivation behavior of S. aureus was closely related to the variables of process pressure, holding-pressure time (HPT), process temperature and process cycling. The log reduction of S. aureus at 40 MPa for 30-min HPT was significantly greater (P < 0.05), but the inactivation effect at 10, 20 and 30 MPa was similar. The log reduction of S. aureus at 30 and 40 MPa for 60-min HPT was similar and significantly greater (P < 0.05), while the inactivation effect at 10 and 20 MPa was similar. The inactivation of S. aureus against HPT conformed to a fast,slow biphase kinetics; the two stages were well fitted to a first-order model with higher regression coefficients R2 = 1.000 and 0.9238; their respective D values (decimal reduction time) were 16.52 and 70.42 min. As the process temperature increased, the log reduction of S. aureus increased significantly (P < 0.05); the inactivation kinetics of S. aureus versus process temperature was characterized with a fast inactivation rate from 32 to 45C and a slow inactivation rate from 45 to 55C. As compared to one-process cycling for a total of 60-min HPT, four-process cycling resulted in a significant reduction of S. aureus, and its maximal reduction was near to 5 log cycles, indicating that more process cycling caused more inactivation of S. aureus under identical pressure and temperature with equal HPT. However, the maximal reduction was 0.09 and 0.12 log cycles for two- and four-process cyclings with 0-min HPT, indicating that pressurization and depressurization had a lesser effect on the inactivation of S. aureus, while HPT was significant in DPCD to inactivate S. aureus. PRACTICAL APPLICATIONS Dense-phase carbon dioxide (DPCD) is a novel technology to achieve cold pasteurization and/or sterilization of liquid and solid materials, and is likely to replace or partially substitute currently and widely applied thermal processes. This study showed that DPCD effectively inactivated Staphylococcus aureus inoculated in 7.5% sodium chloride broth, and the inactivation behavior of S. aureus was closely related to the pressure, holding-pressure time, temperature and process cycling. Based on this observation, the technology of DPCD can be applied in the pasteurization of foods such as milk and various fruit juices, especially thermal-sensitive materials. [source] WATER ACTIVITY AND THE INACTIVATION OF ENTEROBACTER CLOACAE INOCULATED IN CHOCOLATE LIQUOR AND A MODEL SYSTEM BY PULSED ELECTRIC FIELD TREATMENTJOURNAL OF FOOD PROCESSING AND PRESERVATION, Issue 5 2002S. MI Effects of water activity (aw) on the inactivation of Enterobacter cloacae inoculated in chocolate liquor and in a model system of 0.1% (w/v) peptone water and glycerol by pulsed electric field (PEF) treatment were investigated. An electric field strength of 24.5 kV/cm, a total treatment time of 320 ,s, a pulse duration time of 4 ,s, a pulse delay time of 15 ,s, and a pulse cycle time of 15 s were selected for PEF treatment. The inactivation ofE. cloacae by PEF increased significantly as aw increased (P < 0. 05). As aw of chocolate liquor increased from 0.48 to 0.89, the log reduction of E. cloacae increased from 0.1 to 1.3. The measured temperature change inside the PEF treatment chamber was 0.4C when the log reduction was 1. 3. Similarly, as aw increased from 0. 51 to 0.91 in the model system, the log reduction increased from 0.4 to 1.3. E. cloacae surviving a low aw environment had high resistance to PEF. PEF inactivated E. cloacae in the chocolate liquor with aw of 0.85 by 1 log at O h incubation. However, the log reduction was only 0.1 when PEF treatment was applied to E. cloacae which was incubated for 2 h in the chocolate liquor with aw of 0.85 before PEF treatment. E. cloacae surviving the low aw environment might have resistance not only to the low aw but also to PEF. The resistance to low aw environment may need to be considered when the inactivation of microorganisms by PEF is evaluated. [source] A PREDICTIVE MODEL FOR HIGH-PRESSURE CARBON DIOXIDE INACTIVATION OF MICROORGANISMSJOURNAL OF FOOD SAFETY, Issue 2 2009S. BUZRUL ABSTRACT The Weibull model, which is commonly used for thermal inactivation of microorganisms in literature, was used to describe microbial inactivation by high-pressure carbon dioxide (HPCD). The number of parameters of the model was reduced from two to one in order to avoid interrelationship of these parameters with a slight loss of goodness-of-fit. A second-order polynomial function fulfilling a number of constraints was proposed for the secondary modeling of the time-constant parameter of the reduced Weibull model. This function consists of both pressure and temperature and therefore can be used for HPCD treatments. PRACTICAL APPLICATIONS The application of any new technology in food preservation requires a reliable model that accurately describes and predicts the inactivation data of microorganisms. In principle, the methodology presented here could be used to describe and predict the survival data for high-pressure carbon dioxide inactivation of microorganisms at least for some pressure and temperature ranges if the isobaric/isothermal survival curves of these microorganisms are linear, concave upward or downward. [source] INACTIVATION OF BACTERIAL SPORES BY COMBINED ACTION OF HYDROSTATIC PRESSURE AND BACTERIOCINS IN ROAST BEEFJOURNAL OF FOOD SAFETY, Issue 4 2003N. KALCHAYANAND ABSTRACT Foodborne bacterial spores are normally resistant to high hydrostatic pressure; however, at moderate pressure, they can be induced to germinate and outgrow. At this stage, they can be killed by bacteriocin-based biopreservatives (BP-containing pediocin and nisin at 3:7 ratio; BPX, BP + 100 ,g/mL lysozyme; BPY, BPX+ 500 ,g/mL Na-EDTA). Based on this principle, spores of the meat spoilage organism, Clostridium laramie (1,2 × 102 spores/bag) alone or a mixture of four clostridial spores (5 × 103 spores/bag), Clostridium sporogenes, Clostridium perfringens, Clostridium tertium, and Clostridium laramie, were inoculated in roast beef in the presence of 5000 AU/g of bacteriocin-based biopreservatives. The roast beef samples were subjected to hydrostatic pressure (HP) at 345 MPa for 5 min at 60C and stored at 4 or 12C for 84 days or at 25C for 7 days. The HP treatment of roast beef samples inoculated with a mixture of clostridial spores could be stored for 42 days at 4C. The HP in combination with either BPX or BPY extended the shelf-life of roast beef up to 7 days at 25C. The combined treatment of HP and BP controlled the growth of C. laramie spores and extended the shelf-life of roast beef for 84 days when stored at 4C. [source] EFFECT OF MARINADE AND DRYING TEMPERATURE ON INACTIVATION OF ESCHERICHIA COLI O157:H7 ON INOCULATED HOME DRIED BEEF JERKYJOURNAL OF FOOD SAFETY, Issue 3 2002SUSAN N. ALBRIGHT ABSTRACT Beef slices were inoculated (5.7,7.5 log CFU/cm2) with a 4-strain composite of E. coli O157:H7, stored (4C, 24 h), marinated (4C, 24 h), dried for 10 h at 62.5C or 68.3C, and stored for 90 days at 21C. Unmarinated beef slices dried for 10 h at 62.5C were used to determine the relative contribution of the marinate versus temperature treatment in the 62.5C trials. Samples were analyzed (bacterial enumeration with selective and nonselective agar media, pH, and aw) following inoculation, marinating, at 4, 6, 8 and 10 h of drying, and after 30, 60 and 90 days of storage. Marination resulted in slight changes in bacterial populations (,0.3 to + 0.6 log CFU/cm2), but did not enhance bacterial reduction during drying. For all treatments, most bacterial reductions occurred in the first 4 h of drying, with little reduction thereafter. After 10 h of drying, bacterial reductions were 3.2,3.4 log CFU/cm2 for unmarinated beef slices dried at 62.5C. Reductions of 2.2 and 3.0,4.6 log CFU/cm2 were achieved in marinated jerky slices dried at 62.5C and 68.3C, respectively. No treatment resulted in the recommended 5-log reduction at the end of 10 h drying. However, bacteria did become undetectable by direct plating (<10 CFU/cm2) following 30 days of storage in all treatments except the unmarinated beef slices plated on tryptic soy agar (TSA). Additional work is needed to develop procedures for adequate destruction of E. coli O157:H7 during drying of beef jerky. [source] INACTIVATION OF ESCHERICHIA COLI O157:H7 ON INOCULATED ALFALFA SEEDS WITH OZONATED WATER UNDER PRESSURE,JOURNAL OF FOOD SAFETY, Issue 2 2002RATNA R. SHARMA ABSTRACT Alfalfa seeds inoculated with Escherichia coli O157:H7 (,105 CFU/g) were subjected to low hydrostatic pressure. Seeds immersed in ozonated water at 4C were held at 8 and 12-psi ozone pressure for 2, 4, 8, 16, 32, and 64 min. Alternatively, seeds were continuously sparged with ozone for up to 64 min and then held at 12 psi for 5 min. Controls consisted of sparging and pressurization with air. Thirty-two minute treatments of continuous ozone sparging followed by pressurization of seeds at 12 psi for 5 min were repeated with the addition of four surfactants (Tween 20, Tween 80, SPAN 20, and SPAN 80) in the treatment water. Enumeration of E. coli O157:H7 on treated, untreated, and control seeds was done on tryptic soy agar supplemented with 50 ,g/mL of nalidixic acid. The reduction in population of E. coli O157:H7 on seeds treated with the 8 and 12 psi hydrostatic pressure in ozonated water ranged from 0.74 ,1.56 log10 CFU/g and 0.72 , 1.62 log10 CFU/g, respectively. Control treatments carried out with air pressurization of seeds resulted in maximum population reductions of 1.55 log10 and 1.83 log10 CFU/g for 8 and 12 psi, respectively. For seeds treated with continuous ozone sparging (2 , 64 min) followed by pressurization at 12 psi for 5 min, the highest reduction was 2.03 log10 CFU/g. Reductions were, however, not significantly different (P > 0.05) from control treatment (with air) which reduced the populations by 0.57 , 2.19 log10 CFU/g. The presence of surfactants during continuous sparging of water followed by pressurization at 12 psi was not beneficial. None of the treatments adversely affected the germination of the seeds. [source] Decreased Tear Expression with an Abnormal Schirmer's Test Following Botulinum Toxin Type A for the Treatment of Lateral Canthal RhytidesDERMATOLOGIC SURGERY, Issue 2 2002Seth L. Matarasso MD background. Inactivation of muscles of facial expression by chemodenervation with botulinum toxin remains an off-label indication. Nevertheless, it continues to be a safe and effective technique to improve dynamic rhytides and is the treatment of choice for the hypertrophic lateral fibers of the orbicularis oculi muscle that can cause the superimposed crow's feet. objective. Although infrequent and self-limiting, the complication of unexpected muscle weakness from toxin diffusion or erroneous placement is documented. methods. However, injection into the pretarsal portion of the orbicularis oculi muscle resulting in unilateral ocular irritation and diminished tear expression as evidenced by a dry eye and an abnormal Schirmer's test has rarely been reported. Direct injection into the pretarsal fibers of the muscle as opposed to diffusion of the toxin into the muscle fibers or the lacrimal gland was consistent with the onset of action of the toxin and the prolonged duration of the ocular symptoms. results. Treatment consisted of ocular lubrication until the effects of the toxin dissipated and muscle tone returned. Subsequent treatment did not result in a result in a recurrence of adverse sequelae. conclusions. Facial muscles are small, not isolated, and often have fibers that interdigitate. An important factor in the administration of botulinum toxin is the identification of the muscles responsible for the corresponding rhytide. Precise knowledge of muscular anatomy and function will aid in minimizing this and other potential complications. [source] Conditional gene inactivation reveals roles for Fgf10 and Fgfr2 in establishing a normal pattern of epithelial branching in the mouse lungDEVELOPMENTAL DYNAMICS, Issue 8 2009Lisa L. Abler Abstract Fibroblast growth factor 10 (FGF10) signaling through FGF receptor 2 (FGFR2) is required for lung initiation. While studies indicate that Fgf10 and Fgfr2 are also important at later stages of lung development, their roles in early branching events remain unclear. We addressed this question through conditional inactivation of both genes in mouse subsequent to lung initiation. Inactivation of Fgf10 in lung mesenchyme resulted in smaller lobes with a reduced number of branches. Inactivation of Fgfr2 in lung epithelium resulted in disruption of lobes and small epithelial outgrowths that arose arbitrarily along the main bronchi. In both mutants, there was an increase in cell death. Also, the expression patterns of key signaling molecules implicated in branching morphogenesis were altered and a proximal lung marker was expanded distally. Our results indicate that both Fgf10 and Fgfr2 are required for a normal branching program and for proper proximal,distal patterning of the lung.Developmental Dynamics 238:1999,2013, 2009. © 2009 Wiley-Liss, Inc. [source] Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junctionDEVELOPMENTAL NEUROBIOLOGY, Issue 10 2006Thomas Rival Abstract L -Glutamate is the major excitatory neurotransmitter in the mammalian brain. Specific proteins, the Na+/K+ -dependent high affinity excitatory amino acid transporters (EAATs), are involved in the extracellular clearance and recycling of this amino acid. Type I synapses of the Drosophila neuromuscular junction (NMJ) similarly use L -glutamate as an excitatory transmitter. However, the localization and function of the only high-affinity glutamate reuptake transporter in Drosophila, dEAAT1, at the NMJ was unknown. Using a specific antibody and transgenic strains, we observed that dEAAT1 is present at the adult, but surprisingly not at embryonic and larval NMJ, suggesting a physiological maturation of the junction during metamorphosis. We found that dEAAT1 is not localized in motor neurons but in glial extensions that closely follow motor axons to the adult NMJ. Inactivation of the dEAAT1 gene by RNA interference generated viable adult flies that were able to walk but were flight-defective. Electrophysiological recordings of the thoracic dorso-lateral NMJ were performed in adult dEAAT1-deficient flies. The lack of dEAAT1 prolonged the duration of the individual responses to motor nerve stimulation and this effect was progressively increased during physiological trains of stimulations. Therefore, glutamate reuptake by glial cells is required to ensure normal activity of the Drosophila NMJ, but only in adult flies. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Accelerating the secondary immune response by inactivating CD4+CD25+ T regulatory cells prior to BCG vaccination does not enhance protection against tuberculosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2008Kylie M. Quinn Abstract CD4+CD25+ natural T regulatory cells (Tregs) have been shown to suppress protective immune responses in several different vaccination models. Since the effect of Tregs on vaccination against tuberculosis (Tb) was unknown, we used a murine model to investigate whether natural Tregs suppress the development of protective immunity following Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. Using a monoclonal antibody against CD25, natural Tregs were inactivated prior to vaccination with BCG. The primary immune response was evaluated after BCG vaccination and the secondary immune response was assessed after an intranasal BCG challenge 42,days after vaccination. Inactivation of natural Tregs prior to vaccination led to an increased immune response 14,days after vaccination, increased numbers of antigen-responsive lymphocytes immediately prior to secondary challenge and the earlier appearance of IFN-,-producing CD4+ and CD8+ lymphocytes in the draining lymph nodes and lungs after challenge. Despite this, protection from virulent Mycobacterium tuberculosis or M. bovis aerosol challenge was unaffected by natural Treg inactivation prior to BCG vaccination. This suggests that increasing the primary and accelerating the secondary immune responses by inactivating natural Tregs at the time of vaccination, does not affect the development of protective immunity to Tb. [source] Mechanism-Based Inactivation of Coenzyme B12 -Dependent 2-Methyleneglutarate Mutase by (Z)-Glutaconate and Buta-1,3-diene-2,3-dicarboxylateEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 18 2006Wolfgang Buckel Abstract In the presence of holo 2-methyleneglutarate mutase, buta-1,3-diene-2,3-dicarboxylate and (Z)-glutaconate [(Z)-pent-2-ene-1,5-dicarboxylate], but not (E)-glutaconate, each induced homolysis of the Co,C bond of coenzyme B12 to afford cob(II)alamin and the 5,-deoxyadenosyl radical. The latter probably added to the double bond in (Z)-glutaconate and one of the double bonds in buta-1,3-diene-2,3-dicarboxylate to afford a corresponding "radical adduct". The formation of new radicals and cob(II)alamin was diagnosed by UV/Visible and EPR spectroscopy. (Z)-Glutaconate rapidly inactivated the mutase with formation of aquocobalamin, which was possibly derived by electron transfer from cob(II)alamin to the radical adduct. In contrast, buta-1,3-diene-2,3-dicarboxylate was a much slower inactivator. In this case, the spectroscopic data revealed a relatively stable complex of the radical adduct with cob(II)alamin in the active site of the enzyme. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Inactivation of astroglial NF-,B promotes survival of retinal neurons following ischemic injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2009Galina Dvoriantchikova Abstract Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF-,B, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia-reperfusion in wild-type (WT) and in GFAP-I,B,-dn transgenic mice, where the NF-,B classical pathway is suppressed specifically in astrocytes. The GFAP-I,B,-dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro-inflammatory genes, encoding Tnf-,, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO-synthase in the retinas of GFAP-I,B,-dn mice. These data suggest that certain NF-,B-regulated pro-inflammatory and redox-active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke. [source] Inactivation of the gene for the nuclear receptor tailless in the brain preserving its function in the eyeEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2007Thorsten Belz Abstract During embryogenesis, tailless, an orphan member of the nuclear receptor family, is expressed in the germinal zones of the brain and the developing retina, and is involved in regulating the cell cycle of progenitor cells. Consequently, a deletion of the tailless gene leads to decreased cell number with associated anatomical defects in the limbic system, the cortex and the eye. These structural abnormalities are associated with blindness, increased aggressiveness, poor performance in learning paradigms and reduced anxiousness. In order to assess the contribution of blindness to the behavioural changes, we established tailless mutant mice with intact visual abilities. We generated a mouse line in which the second exon of the tailless gene is flanked by loxP sites and crossed these animals with a transgenic line expressing the Cre recombinase in the neurogenic area of the developing brain, but not in the eye. The resulting animals have anatomically indistinguishable brains compared with tailless germline mutants, but are not blind. They are less anxious and much more aggressive than controls, like tailless germline mutants. In contrast to germline mutants, the conditional mutants are not impaired in fear conditioning. Furthermore, they show good performance in the Morris water-maze despite severely reduced hippocampal structures. Thus, the pathological aggressiveness and reduced anxiety found in tailless germline mutants are due to malformations caused by inactivation of the tailless gene in the brain, but the poor performance of tailless null mice in learning and memory paradigms is dependent on the associated blindness. [source] Axon behaviour at Schwann cell , astrocyte boundaries: manipulation of axon signalling pathways and the neural adhesion molecule L1 can enable axons to crossEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2004Kathryn H. Adcock Abstract Axon regeneration in vivo is blocked at boundaries between Schwann cells and astrocytes, such as occur at the dorsal root entry zone and around peripheral nerve or Schwann cell grafts. We have created a tissue culture model of these boundaries in Schwann cell , astrocyte monolayer co-cultures. Axon behaviour resembles that in vivo, with axons showing a strong preference for Schwann cells over astrocytes. At boundaries between the two cell types, axons growing on astrocytes cross readily onto Schwann cells, but only 15% of axons growing on Schwann cells are able to cross onto astrocytes. Treatment with chondroitinase or chlorate to reduce inhibition by proteoglycans did not change this behaviour. The neural adhesion molecule L1 is present on Schwann cells and not astrocytes, and manipulation of L1 by application of an antibody, L1-Fc in solution, or adenoviral transduction of L1 into astrocytes increased the proportion of axons able to cross onto astrocytes to 40,50%. Elevating cAMP levels increased crossing from Schwann cells onto astrocytes in live and fixed cultures, and had a co-operative effect with NT-3 but not with NGF. Inactivation of Rho with a cell-permeant form of C3 exoenzyme also increased crossing from Schwann cells to astrocytes. Our experiments indicate that the preference of axons for Schwann cells is largely mediated by the presence of L1 on Schwann cells but not astrocytes, and that manipulation of growth cone signalling pathways can allow axons to disregard boundaries between the two cell types. [source] Otx1 gene-controlled morphogenesis of the horizontal semicircular canal and the origin of the gnathostome characteristicsEVOLUTION AND DEVELOPMENT, Issue 4 2000Sylvie Mazan SUMMARY The horizontal semicircular canal of the inner ear is a unique feature of gnathostomes and is predated by the two vertical semicircular canals, which are already present in lampreys and some fossil, armored jawless vertebrates regarded as close relatives of gnathostomes. Inactivation in mice of the orthodenticle -related gene Otx1 results in the absence of this structure. In bony fishes and tetrapods (osteichthyans), this gene belongs to a small multigene family comprising at least two orthology classes, Otx1 and Otx2. We report that, as in the mouse, xenopus and zebrafish, Otx1- and Otx2 -related genes are present in a chondrichthyan, the dogfish Scyliorhinus canicula, with an Otx1 expression domain in the otocyst very similar to those observed in osteichthyans. A strong correlation is thus observed in extant vertebrates between the distribution of the horizontal semicircular canal and the presence of an Otx1 ortholog expressed in the inner ear, which supports the hypothesis that the absence of this characteristic in Otx1 -/- mice may correspond to an atavism. The same conclusion applies to two other gnathostome-specific characteristics also deleted in Otx1 -/- mice, the utriculosaccular duct and the ciliary process. Together with functional analyses of Otx1 and Otx2 genes in mice and comparative analyses of the Otx gene families characterized in chordates, these discoveries lead to the hypothesis that some of the anatomic characteristics of gnathostomes have appeared quite suddenly and almost simultaneously in vertebrate evolution, possibly as a consequence of gene functional diversifications following duplications of an ancestral chordate gene. [source] Inactivation of colicin Y by intramembrane helix,helix interaction with its immunity proteinFEBS JOURNAL, Issue 21 2008David, majs The construction of hybrids between colicins U and Y and the mutagenesis of the colicin Y gene (cya) have revealed amino acid residues important for interactions between colicin Y and its cognate immunity protein (Cyi). Four such residues (I578, T582, Y586 and V590) were found in helices 8 and 9 of the colicin Y pore-forming domain. To verify the importance of these residues, the corresponding amino acids in the colicin B protein were mutated to the residues present in colicin Y. An Escherichia coli strain with cloned colicin Y immunity gene (cyi) inactivated this mutant, but not the wild-type colicin B. In addition, interacting amino acid pairs in Cya and Cyi were identified using a set of Cyi point mutant strains. These data are consistent with antiparallel helix,helix interactions between Cyi helix T3 and Cya helix 8 of the pore-forming domain as a molecular mechanism of colicin Y inactivation by its immunity protein. [source] Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesisFEBS JOURNAL, Issue 13 2003Susan Aiston Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis. [source] Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoidesFEBS JOURNAL, Issue 22 2002Nicolas Sauvageot The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a ,2,2,2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 µm. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 °C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway. [source] Inactivation of Aeromonas hydrophila metallo-,-lactamase by cephamycins and moxalactamFEBS JOURNAL, Issue 13 2001Astrid Zervosen Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-,-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3, leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate. [source] Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesisFEBS JOURNAL, Issue 22 2000Nikolas E. Labrou The 2,,3,-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min,1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme,oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme,oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+ -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360,Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360,Ala enzyme. The data are discussed in terms of engineering coenzyme specificity. [source] Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxideFEBS JOURNAL, Issue 5 2000Evidence for a dithiol, disulfide equilibrium, implications for redox regulation Calcineurin (CaN) is a Ca2+ -and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe,Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3,8 µm and the inactivation was reversed by 2,3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+,Zn2+ dinuclear centre. Upon H2O2 -mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN. [source] Inactivation of pqq genes of Enterobacter intermedium 60-2G reduces antifungal activity and induction of systemic resistanceFEMS MICROBIOLOGY LETTERS, Issue 1 2008Song Hee Han Abstract Enterobacter intermedium 60-2G, a phosphate solubilizing bacterium, has the ability to induce systemic resistance in plants against soft rot pathogen Erwinia carotovora. Glucose dehydrogenase, an enzyme that utilizes pyrroloquinoline quinone (PQQ) as a cofactor, is required for the synthesis of gluconic acid by E. intermedium 60-2G. Here, we report that the pqqA and pqqB genes are required for phosphate solubilization and induced systemic resistance against a soft rot pathogen in tobacco. Mutations in either the pqqA or pqqB gene abolished the production of 2-ketogluconic acid and eliminated the ability of E. intermedium to solubilize hydroxyapatite. Addition of gluconic acid to the growth media restored the ability of the pqqA mutant to produce 2-ketogluconic acid. Interestingly, both pqqA and pqqB mutants of E. intermedium lost their ability to inhibit the growth of the rice pathogen Magnaporthe grisea KI-409. Additionally, induced systemic resistance against the soft rot pathogen was attenuated in the pqq mutants. These functions were restored by complementation with the wild-type pqq gene cluster. Our findings suggest that PQQ plays an important function in beneficial traits including phosphate solubilization, antifungal activity, and induced systemic resistance of E. intermedium, possibly by acting as a cofactor for several enzymes including glucose dehydrogenase. [source] RegM is required for optimal fructosyltransferase and glucosyltransferase gene expression in Streptococcus mutansFEMS MICROBIOLOGY LETTERS, Issue 1 2004Christopher M. Browngardt Abstract Glucosyltransferases (Gtfs) and fructosyltransferase (Ftf), and the exopolysaccharides they produce, facilitate bacterial adherence and biofilm formation, and enhance the virulence of Streptococcus mutans. In this study, we used continuous chemostat cultures and reporter gene fusions to study the expression of ftf and gtfBC in response to carbohydrate availability and pH, and to asses the role of a protein similar to catabolite control protein A (CcpA), RegM, in regulation of these genes. Expression of ftf was efficient at pH 7.0 and 6.0, but was repressed at pH 5.0 under glucose-excess conditions. At pH 7.0, ftf expression was 5-fold lower under glucose-limiting conditions than in cells growing with an excess of glucose. Expression of gtfBC was also sensitive, albeit to a lesser extent, to pH and glucose availability. Inactivation of regM resulted in decreases of as much as 10-fold in both ftf and gtfBC expression, depending on growth conditions. These findings reinforce the importance of pH and carbohydrate availability for expression of two primary virulence attributes of S. mutans and reveal a critical role for RegM in regulation of expression of both gtfBC and ftf. [source] Inactivation of the cystatin E/M tumor suppressor gene in cervical cancerGENES, CHROMOSOMES AND CANCER, Issue 9 2008Mysore S. Veena We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5,aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation. © 2008 Wiley-Liss, Inc. [source] Analysis of somatic APC mutations in rare extracolonic tumors of patients with familial adenomatous polyposis coliGENES, CHROMOSOMES AND CANCER, Issue 2 2004Hendrik Bläker Patients with familial adenomatous polyposis coli (FAP) carry heterozygous mutations of the APC gene. At a young age, these patients develop multiple colorectal adenomas that consistently display a second somatic mutation in the remaining APC wild-type allele. Inactivation of APC leads to impaired degradation of ,-catenin, thereby promoting continuous cell-cycle progression. The role of APC inactivation in rare extracolonic tumors of FAP patients has not been characterized sufficiently. Among tissue specimen from 174 patients with known APC germ-line mutations, we identified 8 tumors infrequently seen in FAP. To investigate the pathogenic role of APC pathway deregulation in these lesions, they were analyzed for second-hit somatic mutations in the mutational cluster region of the APC gene. Immunohistochemistry was performed to compare the expression pattern of ,-catenin to the mutational status of the APC gene. Exon 3 of the ,-catenin gene (CTNNB1) was analyzed for activating mutations to investigate alternative mechanisms of elevated ,-catenin concentration. Although CTNNB1 mutations were not observed, second somatic APC mutations were found in 4 of the 8 tumors: a uterine adenocarcinoma, a hepatocellular adenoma, an adrenocortical adenoma, and an epidermal cyst. These tumors showed an elevated concentration of ,-catenin. No APC mutations were seen in focal nodular hyperplasia of the liver, angiofibrolipoma, and seborrheic wart. This is the first study reporting second somatic APC mutations in FAP-associated uterine adenocarcinoma and epidermal cysts. Furthermore, our data strengthen a role for impaired APC function in the pathogenesis of adrenal and hepatic neoplasms in FAP patients. © 2004 Wiley-Liss, Inc. [source] Abnormal lens morphogenesis and ectopic lens formation in the absence of ,-catenin function,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2007Jana Kreslova Abstract ,-Catenin plays a key role in cadherin-mediated cell adhesion as well as in canonical Wnt signaling. To study the role of ,-catenin during eye development, we used conditional Cre/loxP system in mouse to inactivate ,-catenin in developing lens and retina. Inactivation of ,-catenin does not suppress lens fate, but instead results in abnormal morphogenesis of the lens. Using BAT-gal reporter mice, we show that ,-catenin-mediated Wnt signaling is notably absent from lens and neuroretina throughout eye development. The observed defect is therefore likely due to the cytoskeletal role of ,-catenin, and is accompanied by impaired epithelial cell adhesion. In contrast, inactivation of ,-catenin in the nasal ectoderm, an area with active Wnt signaling, results in formation of crystallin-positive ectopic lentoid bodies. These data suggest that, outside of the normal lens, ,-catenin functions as a coactivator of canonical Wnt signaling to suppress lens fate. genesis 45:157,168, 2007. Published 2007 Wiley-Liss, Inc. [source] Inactivation of oxidized and S -nitrosylated mitochondrial proteins in alcoholic fatty liver of rats,HEPATOLOGY, Issue 5 2006Kwan-Hoon Moon Increased oxidative/nitrosative stress is a major contributing factor to alcohol-mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol-induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S -nitrosylated mitochondrial proteins and to use a biotin- N -maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol-inducible CYP2E1. The biotin- N -maleimide-labeled oxidized and/or S -nitrosylated mitochondrial proteins from pair-fed controls or alcohol-fed rat livers were subsequently purified with streptavidin-agarose. The overall patterns of oxidized and/or S -nitrosylated proteins resolved by 2-dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy-nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3-ketoacyl-CoA thiolase involved in mitochondrial ,-oxidation of fatty acids was significantly inhibited in alcohol-exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion, our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol-mediated liver damage. (HEPATOLOGY 2006;44:1218,1230.) [source] | ||||||||||||||||||||||||||||||||||||||||||||||