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Absolute Requirement (absolute + requirement)
Selected AbstractsDendritic cell-derived IL-15 controls the induction of CD8 T,cell immune responsesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2003René Rückert Abstract The development and the differentiation of CD8+ T,cells are dependent on IL-15. Here, we have studied the source and mechanism of how IL-15 modulates CD8+ T,cell-mediated Th1immune responses by employing two delayed-type hypersensitivity (DTH) models. IL-15-deficient (IL-15,/,) mice or mice treated with soluble IL-15R, as an IL-15 antagonist showed significantly reduced CD8+ T,cell-dependent DTH responses, while activation of CD4+ T,cell and B,cell functions remained unaffected. Injection of antigen-labeled dendritic cells (DC) fromIL-15+/+, IL-15,/, or IL-15R,,/, mice revealed that DC-derived IL-15 is an absolute requirement for the initiation of DTH response. The re-establishment of the interaction of IL-15 with the IL-15R, by incubating IL-15,/, DC with IL-15 completely restored the capacity to prime T,cells for DTH induction in vivo. Moreover, IL-15 also enhanced secretion of pro-inflammatory cytokines by DC and triggered in vitro CD8+ T,cell proliferation and IL-2 release. Taken together, the data suggest that an autocrine IL-15/IL-15R, signaling loop in DC is essential for inducing CD8+ -dependent Th1 immune responses in mice. Therefore, targeted manipulation of this loop promises to be an effective, novel strategy for therapeuticmodulation of clinically relevant DTH reactions. [source] Isoprenoid biosynthesis in plants , 2C -methyl- d -erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thalianaFEBS JOURNAL, Issue 19 2006Felix Rohdich The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C -methyl- d -erythritol 4-phosphate from 1-deoxy- d -xylulose 5-phosphate at a rate of 5.6 µmol·min,1·mg,1 (kcat 4.4 s,1). The Michaelis constants for 1-deoxy- d -xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 µm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 µmol·min -1·mg -1. [source] Schwann cell myelination occurred without basal lamina formation in laminin ,2 chain-null mutant (dy3K/dy3K) miceGLIA, Issue 2 2001Masahiro Nakagawa Abstract The laminin ,2 chain is a major component of basal lamina in both skeletal muscle and the peripheral nervous system. Laminin ,2 chain deficiency causes merosin-deficient congenital muscular dystrophy, which affects not only skeletal muscles, but also the peripheral and central nervous systems. It has been reported that the formation of basal lamina is required for myelination in the peripheral nervous system. In fact, the spinal root of dystrophic mice (dy/dy mice), whose laminin ,2 chain expression is greatly reduced, shows lack of basal lamina and clusters of naked axons. To investigate the role of laminin ,2 chain and basal lamina in vivo, we examined the peripheral nervous system of dy3K/dy3Kmice, which are null mutants of laminin ,2 chain. The results indicate the presence of myelination although Schwann cells lacked basal lamina in the spinal roots of dy3K/dy3K mice, suggesting that basal lamina is not an absolute requirement for myelination in vivo. Immunohistochemically, the expression of laminin ,4 chain was increased and laminin ,5 chain was preserved in the endoneurium of the spinal root. Laminin ,4 and ,5 chains may play the critical role in myelination instead of laminin ,2 chain in dy3K/dy3Kmice. In addition, the motor conduction velocity of the sciatic nerve was significantly reduced compared with that of wild-type littermate. This reduction in conduction velocity may be due to small axon diameter, thin myelin sheath and the patchy disruption of the basal lamina of the nodes of Ranvier in dy3K/dy3Kmice. GLIA 35:101,110, 2001. © 2001 Wiley-Liss, Inc. [source] Is Helicobacter pylori a True Microaerophile?HELICOBACTER, Issue 4 2006Stephanie Bury-Moné Abstract Background:, There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5,19% and carbon dioxide tensions 5,10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. Methods:, Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. Results:,H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO2, the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (< 5%) to fully aerobic (21%) at cell densities higher than 5 × 105 cfu/ml for media supplemented with horse serum and 5 × 107 cfu/ml for media supplemented with ,-cyclodextrin. Evidence that changes occurred in the physiology of H. pylori was obtained by comparing the activities of ferredoxin:NADH (nicotinamide adenine dinucleotide) oxidoreductases of bacteria grown under microaerobic and aerobic atmospheres. Conclusions:,H. pylori is a capnophile able to grow equally well in vitro under microaerobic or aerobic conditions at high bacterial concentrations, and behaved like oxygen-sensitive microaerophiles at low cell densities. Some characteristics of H. pylori cells grown in vitro under microaerobic conditions appeared to mimic better the physiology of organisms grown in their natural niche in the human stomach. [source] Effects of aromatic amino acids, phenylacetate and phenylpropionate on fermentation of xylan by the rumen anaerobic fungi, Neocallimastix frontalis and Piromyces communisJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007A.Y. Guliye Abstract Aims:, Anaerobic fungi are important members of the fibrolytic community of the rumen. The aim of this study was to study their requirement for aromatic amino acids (AA) and related phenyl acids (phenylpropionic and phenylacetic acids) for optimal xylan fermentation. Methods and Results:,Neocallimastix frontalis RE1 and Piromyces communis P were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia; ammonia plus a complete mixture of 20 AA commonly found in protein; ammonia plus complete AA mixture minus aromatic AA; ammonia plus phenyl acids; ammonia plus complete AA mixture without aromatic AA plus phenyl acids. Both species grew in all the media, indicating no absolute requirement for AA. The complete AA mixture increased (P < 0·05) acetate concentration by 18% and 15%, sugar utilization by 33% and 22% and microbial yield by about 22% and 15% in N. frontalis and P. communis, respectively, in comparison with the treatments that had ammonia as the only N source. Neither the supply of aromatic AA or phenol acids, nor their deletion from the complete AA mixture, affected the fermentation rate, products or yield of either species. Conclusions:, AA were not essential for N. frontalis and P. communis, but their growth on xylan was stimulated. The effects could not be explained in terms of aromatic AA alone. Significance and Impact of the Study:, Ruminant diets should contain sufficient protein to sustain optimal fibre digestion by ruminal fungi. Aromatic AA or phenyl acids alone cannot replace the complete AA mixture. [source] Auxotrophic mutant of the cyanobacterium Nostoc muscorum showing absolute requirement of Cs+ or Rb+ for diazotrophy and autotrophyJOURNAL OF BASIC MICROBIOLOGY, Issue 4 2006Santosh Bhargava Dr. Caesium-resistant (Cs+ -R) mutant clones of the cyanobacterium Nostoc muscorum were characterized for diazotrophic growth in a medium devoid of Cs+ or Rb+ or both. Cs+ -R phenotype suffered severe genetic damage of a pleiotropic nature affecting diazotrophic growth, chlorophyll a content, nitrogenase activity and photosynthetic O2 evolution. Mutation leading to development of Cs+ -R phenotype could be overcome by availability of Cs+/Rb+. Parent and mutant strains were similar with respect to their Cs+/Rb+ uptake. Available data suggests operation of an efficient coupling of the two incompatible reactions viz. oxygenic photosynthesis and oxygen sensitive N2 fixation in this cyanobacterium. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Metyrapone-Induced Glucocorticoid Depletion Modulates Tyrosine Hydroxylase and Phenylethanolamine N -Methyltransferase Gene Expression in the Rat Adrenal Gland by a Noncholinergic Transsynaptic ActivationJOURNAL OF NEUROENDOCRINOLOGY, Issue 1 2003C. Laborie Abstract The hypothalamic corticotropin-releasing hormone system and the sympathetic nervous system are anatomically and functionally interconnected and hormones of the hypothalamic-pituitary-adrenocortical axis contribute to the regulation of catecholaminergic systems. To investigate the role of glucocorticoids on activity of the adrenal gland, we analysed plasma and adrenal catecholamines, tyrosine hydroxylase (TH) and phenylethanolamine N -methyltransferase (PNMT) mRNA expression in rats injected with metyrapone or dexamethasone. Metyrapone-treated rats had significantly lower epinephrine and higher norepinephrine production than control rats. Metyrapone increased TH protein synthesis and TH mRNA expression whereas its administration did not affect PNMT mRNA expression. Dexamethasone restored plasma and adrenal epinephrine concentrations and increased PNMT mRNA levels, which is consistent with an absolute requirement of glucocorticoids for PNMT expression. Adrenal denervation completely abolished the metyrapone-induced TH mRNA expression. Blockage of cholinergic neurotransmission by nicotinic or muscarinic receptor antagonists did not prevent the metyrapone-induced rise in TH mRNA. Finally, pituitary adenylate cyclase activating polypeptide (PACAP) adrenal content was not affected by metyrapone. These results provide evidence that metyrapone-induced corticosterone depletion elicits transsynaptic TH activation, implying noncholinergic neurotransmission. This may involve neuropeptides other than PACAP. [source] Differentiation between electron transport sensing and proton motive force sensing by the Aer and Tsr receptors for aerotaxisMOLECULAR MICROBIOLOGY, Issue 3 2006Jessica C. Edwards Summary Aerotaxis (oxygen-seeking) behaviour in Escherichia coli is a response to changes in the electron transport system and not oxygen per se. Because changes in proton motive force (PMF) are coupled to respiratory electron transport, it is difficult to differentiate between PMF, electron transport or redox, all primary candidates for the signal sensed by the aerotaxis receptors, Aer and Tsr. We constructed electron transport mutants that produced different respiratory H+/e, stoichiometries. These strains expressed binary combinations of one NADH dehydrogenase and one quinol oxidase. We then introduced either an aer or tsr mutation into each mutant to create two sets of electron transport mutants. In vivo H+/e, ratios for strains grown in glycerol medium ranged from 1.46 ± 0.18,3.04 ± 0.47, but rates of respiration and growth were similar. The PMF jump in response to oxygen was proportional to the H+/e, ratio in each set of mutants (r2 = 0.986,0.996). The length of Tsr-mediated aerotaxis responses increased with the PMF jump (r2 = 0.988), but Aer-mediated responses did not correlate with either PMF changes (r2 = 0.297) or the rate of electron transport (r2 = 0.066). Aer-mediated responses were linked to NADH dehydrogenase I, although there was no absolute requirement. The data indicate that Tsr responds to changes in PMF, but strong Aer responses to oxygen are associated with redox changes in NADH dehydrogenase I. [source] A novel substitution I381V in the sterol 14,-demethylase (CYP51) of Mycosphaerella graminicola is differentially selected by azole fungicidesMOLECULAR PLANT PATHOLOGY, Issue 3 2007B. A. FRAAIJE SUMMARY The recent reduction in the efficacy of azole fungicides in controlling Septoria leaf blotch of wheat, caused by Mycosphaerella graminicola, has prompted concerns over possible development of resistance, particularly in light of the recent emergence of widespread resistance to quinone outside inhibitors (QoIs). We have recently implicated alterations in the target-encoding sterol 14,-demethylase protein (CYP51), and over-expression of genes encoding efflux pumps, in reducing sensitivity to the azole class of sterol demethylation inhibitors (DMIs) in M. graminicola. Here we report on the prevalence and selection of two CYP51 alterations, substitution I381V and deletion of codons 459 and 460 (,Y459/G460), in populations of M. graminicola. Neither alteration has previously been identified in human or plant pathogenic fungi resistant to azoles. The presence of ,Y459/G460 showed a continuous distribution of EC50 values across isolates with either I381 or V381, and had no measurable effect on azole sensitivity. Data linking fungicide sensitivity with the presence of I381V in M. graminicola show for the first time that a particular CYP51 alteration is differentially selected by different azoles in field populations of a plant pathogen. Substitution I381V although not an absolute requirement for reduced azole sensitivity, is selected by tebuconazole and difenoconazole treatment, suggesting an adaptive advantage in the presence of these two compounds. Prochloraz treatments appeared to select negatively for I381V, whereas other azole treatments did not or only weakly impacted on the prevalence of this substitution. These findings suggest treatments with different members of the azole class of fungicides could offer a resistance management strategy. [source] Mosquito midguts and malaria: cell biology, compartmentalization and immunologyPARASITE IMMUNOLOGY, Issue 4 2006M. M. A. WHITTEN SUMMARY The malaria parasite Plasmodium has an absolute requirement for both a vertebrate and a mosquito host in order to complete its life cycle, and its interactions with the latter provide the focus for this review. The mosquito midgut represents one of the most challenging environments for the survival and development of Plasmodium, and is thus also one of the most attractive sites for novel targeted malaria control strategies. During their attempts to cross the midgut epithelium en route to the salivary glands, motile ookinetes are swiftly detected and labelled by mosquito recognition factors and targeted for destruction by a variety of immune responses that recruit killing factors both from the midgut and from other tissues in the surrounding body cavity. The exact interplay between these factors and the parasite is highly species- and strain-specific, as are the timing and the route of parasite invasion. These features are paramount to determining the success of the infection and the vector competence of the mosquito. Here we discuss recent advances in genomic analyses, coupled with detailed microscopical investigations, which are helping to unravel the identity and roles of the major players of these complex systems. [source] Conditioning period, CO2 and GR24 influence ethylene biosynthesis and germination of Striga hermonthicaPHYSIOLOGIA PLANTARUM, Issue 1 2000Abdel Gabbar T. Babiker Germination of witchweed (Striga hermonthica [Del.] Benth), an important root parasite on poaceous crops, requires pretreatment ,conditioning' in a warm moist environment and a subsequent exposure to a stimulant. The roles of conditioning period, CO2 and a strigol analogue (GR24) in ethylene biosynthesis and germination of the parasite were investigated. Conditioning increased the seeds' capacity to oxidize exogenous 1-aminocyclopropane-1-carboxylic acid (ACC). Exogenous CO2 increased the seeds capacity to oxidize ACC by 3- to 9-fold. A combination of GR24 and ACC increased ethylene production by more than 3-fold in comparison with the rates obtained using these compounds separately. Aminoethoxyvinylglycine (AVG) completely inhibited ethylene induction by GR24, but not by ACC. A GR24 treatment, made subsequent to conditioning in GR24, did not induce ethylene. However, seeds conditioned in GR24 and then given 1 mM ACC produced 293 nl l,1 ethylene. ACC oxidase (ACCO) activity in crude extracts was increased by conditioning and CO2. The enzyme displayed an absolute requirement for ascorbate. Absence of exogenous Fe2+ reduced enzyme activity only by 14%. GR24 applied during conditioning reduced germination in response to a subsequent GR24 treatment. ACC was, invariably, less effective in inducing S. hermonthica germination than GR24 even at concentrations which induce more ethylene than concurrent GR24 treatments. The results are consistent with a model in which conditioning removes a restriction on the ethylene biosynthetic pathway in S. hermonthica seeds. GR24 modulates the key enzymes in ethylene biosynthesis. The stimulant suppresses ethylene biosynthesis in unconditioned seeds and promotes it in conditioned ones. Germination of S. hermonthica results from the joint action of GR24 and the ethylene it induces. [source] Structural and biochemical characterization of a novel Mn2+ -dependent phosphodiesterase encoded by the yfcE genePROTEIN SCIENCE, Issue 7 2007Darcie J. Miller Abstract Escherichia coli YfcE belongs to a conserved protein family within the calcineurin-like phosphoesterase superfamily (Pfam00149) that is widely distributed in bacteria and archaea. Superfamily members are metallophosphatases that include monoesterases and diesterases involved in a variety of cellular functions. YfcE exhibited catalytic activity against bis- p -nitrophenyl phosphate, a general substrate for phosphodiesterases, and had an absolute requirement for Mn2+. However, no activity was observed with phosphodiesters and over 50 naturally occurring phosphomonoesters. The crystal structure of the YfcE phosphodiesterase has been determined to 2.25 Ĺ resolution. YfcE has a ,-sandwich architecture similar to metallophosphatases of common ancestral origin. Unlike its more complex homologs that have added structural elements for regulation and substrate recognition, the relatively small 184-amino-acid protein has retained its ancestral simplicity. The tetrameric protein carries two zinc ions per active site from the E. coli extract that reflect the conserved di-Mn2+ active site geometry. A cocrystallized sulfate inhibitor mimics the binding of phosphate moeities in known ligand/phosphatase complexes. Thus, YfcE has a similar active site and biochemical mechanism as well-characterized superfamily members, while the YfcE phosphodiester-containing substrate is unique. [source] Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: Off-pathway aggregation propensity does not determine the co-chaperonin requirementPROTEIN SCIENCE, Issue 12 2000Paul A. Voziyan Abstract One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions. [source] Ion-dependent gating of kainate receptorsTHE JOURNAL OF PHYSIOLOGY, Issue 1 2010Derek Bowie Ligand-gated ion channels are an important class of signalling protein that depend on small chemical neurotransmitters such as acetylcholine, l -glutamate, glycine and ,-aminobutyrate for activation. Although numerous in number, neurotransmitter substances have always been thought to drive the receptor complex into the open state in much the same way and not rely substantially on other factors. However, recent work on kainate-type (KAR) ionotropic glutamate receptors (iGluRs) has identified an exception to this rule. Here, the activation process fails to occur unless external monovalent anions and cations are present. This absolute requirement of ions singles out KARs from all other ligand-gated ion channels, including closely related AMPA- and NMDA-type iGluR family members. The uniqueness of ion-dependent gating has earmarked this feature of KARs as a putative target for the development of selective ligands; a prospect all the more compelling with the recent elucidation of distinct anion and cation binding pockets. Despite these advances, much remains to be resolved. For example, it is still not clear how ion effects on KARs impacts glutamatergic transmission. I conclude by speculating that further analysis of ion-dependent gating may provide clues into how functionally diverse iGluRs families emerged by evolution. Consequently, ion-dependent gating of KARs looks set to continue to be a subject of topical inquiry well into the future. [source] The molecular architecture of the arachidonate-regulated Ca2+ -selective ARC channel is a pentameric assembly of Orai1 and Orai3 subunitsTHE JOURNAL OF PHYSIOLOGY, Issue 17 2009Olivier Mignen The activation of Ca2+ entry is a critical component of agonist-induced cytosolic Ca2+ signals in non-excitable cells. Although a variety of different channels may be involved in such entry, the recent identification of the STIM and Orai proteins has focused attention on the channels in which these proteins play a key role. To date, two distinct highly Ca2+ -selective STIM1-regulated and Orai-based channels have been identified , the store-operated CRAC channels and the store-independent arachidonic acid activated ARC channels. In contrast to the CRAC channels, where the channel pore is composed of only Orai1 subunits, both Orai1 and Orai3 subunits are essential components of the ARC channel pore. Using an approach involving the co-expression of a dominant-negative Orai1 monomer along with different preassembled concatenated Orai1 constructs, we recently demonstrated that the functional CRAC channel pore is formed by a homotetrameric assembly of Orai1 subunits. Here, we use a similar approach to demonstrate that the functional ARC channel pore is a heteropentameric assembly of three Orai1 subunits and two Orai3 subunits. Expression of concatenated pentameric constructs with this stoichiometry results in the appearance of large currents that display all the key biophysical and pharmacological features of the endogenous ARC channels. They also replicate the essential regulatory characteristics of native ARC channels including specific activation by low concentrations of arachidonic acid, complete independence of store depletion, and an absolute requirement for the pool of STIM1 that constitutively resides in the plasma membrane. [source] Magnetic resonance microscopy versus light microscopy in human embryology teachingCLINICAL ANATOMY, Issue 5 2004J. Puerta-Fonollá Abstract A study was carried out on the application of magnetic resonance microscopy (MRM) in teaching prenatal human development. Human embryos measuring 8 mm, 15 mm, 18.5 mm, and 22 mm were fixed in a 4% paraformaldehyde solution and sections obtained with magnetic resonance imaging (MRI) were compared to those prepared for light microscopy (LM), using the same embryos. The MRM and LM slices were of a similar quality. In the MRM sections, embryonic organs and systems were clearly visible, particularly the peripheral and central nervous systems, and the cardiovascular and digestive systems. The digitalization and clarity of the MRM images make them an ideal teaching aid that is suitable for students during the first years of a health-science degree, particularly medicine. As well as providing students with their first experience of MRM, these images allow students to access, at any time, all embryos used, to assess changes in the positions of different organs throughout their stages of development, and to acquire spatial vision, an absolute requirement in the study of human anatomy. We recommend that this technique be incorporated into the wealth of standard embryonic teaching methods already in use. Clin. Anat. 17:429,435, 2004. © 2004 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||