Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation.
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The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment. (+info)
Suppression of inducible nitric oxide synthase by 10-23 DNAzymes in murine macrophage.
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iNOS mRNA of J774 murine macrophage cells was cleaved by 10-23 DNAzymes. DNAzyme target site I or translation initiation site and site II have computer predicted (MFOLD) secondary structures but site III has no secondary structure. All the three DNAzymes cleaved the short transcripts generated from cloned DNA almost with equal efficiency while cleavage efficiency is higher at site III than the other two sites on isolated iNOS mRNA. Interestingly, at intracellular level, DNAzyme targeted at translation initiation codon (site I) having secondary structure cleaved iNOS mRNA, and suppressed its activity and protein expression more efficiently than that targeted at sites II and III. (+info)
Transfection of drug-specific T-cell receptors into hybridoma cells: tools to monitor drug interaction with T-cell receptors and evaluate cross-reactivity to related compounds.
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In the context of drug hypersensitivity, our group has recently proposed a new model based on the structural features of drugs (pharmacological interaction with immune receptors; p-i concept) to explain their recognition by T cells. According to this concept, even chemically inert drugs can stimulate T cells because certain drugs interact in a direct way with T-cell receptors (TCR) and possibly major histocompatibility complex molecules without the need for metabolism and covalent binding to a carrier. In this study, we investigated whether mouse T-cell hybridomas transfected with drug-specific human TCR can be used as an alternative to drug-specific T-cell clones (TCC). Indeed, they behaved like TCC and, in accordance with the p-i concept, the TCR recognize their specific drugs in a direct, processing-independent, and dose-dependent way. The presence of antigen-presenting cells was a prerequisite for interleukin-2 production by the TCR-transfected cells. The analysis of cross-reactivity confirmed the fine specificity of the TCR and also showed that TCR transfectants might provide a tool to evaluate the potential of new drugs to cause hypersensitivity due to cross-reactivity. Recombining the alpha- and beta-chains of sulfanilamide- and quinolone-specific TCR abrogated drug reactivity, suggesting that both original alpha- and beta-chains were involved in drug binding. The TCR-transfected hybridoma system showed that the recognition of two important classes of drugs (sulfanilamides and quinolones) by TCR occurred according to the p-i concept and provides an interesting tool to study drug-TCR interactions and their biological consequences and to evaluate the cross-reactivity potential of new drugs of the same class. (+info)
Evidence for the participation of nitric oxide in pemphigus.
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Pemphigus is an inflammatory autoimmune disorder of the skin. Nitric oxide (NO) is an inflammatory mediator linked to a variety of physiological and pathophysiological phenomena that include skin tumors, psoriasis, urticaria, and atopic dermatitis. Inflammatory cells present in pemphigus lesions are important sources of NO production. We investigated whether NO is involved in pemphigus. A prospective cohort study was conducted at the Dermatology Service of the Hospital Universitario Walter Cantidio of the Federal University of Ceara. All patients seen at the outpatient clinic between August 2000 and July 2001, with a clinically and histologically confirmed diagnosis of pemphigus were included. The median age was 42.5 years (range: 12-69 years) with a male to female ratio of 3:2. Total serum nitrite levels, used as a marker for NO production, were determined by the Griess reaction. Skin biopsies from pemphigus and breast surgery (control) patients were used for the detection of the inducible NO synthase (iNOS) by immunohistochemistry. Twenty-two (22) patients with pemphigus and eight (8) controls who did not differ in demographic characteristics were included. Total serum nitrite levels were significantly higher (>7 micromol/L) in pemphigus patients compared to controls (<6 micromol/L), regardless of the severity of the clinical activity of pemphigus (P < 0.0001). All pemphigus biopsies presented increased immunostaining for iNOS that was not detected in normal skin samples. These data are the first to demonstrate that pemphigus patients display increased serum NO levels that are associated with increased iNOS expression in the affected skin. (+info)
A MORN-repeat protein is a dynamic component of the Toxoplasma gondii cell division apparatus.
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Apicomplexan parasites divide and replicate through a complex process of internal budding. Daughter cells are preformed within the mother on a cytoskeletal scaffold, endowed with a set of organelles whereby in the final stages the mother disintegrates and is recycled in the emerging daughters. How the cytoskeleton and the various endomembrane systems interact in this dynamic process remains poorly understood at the molecular level. Through a random YFP fusion screen we have identified two Toxoplasma gondii proteins carrying multiple membrane occupation and recognition nexus (MORN) motifs. MORN1 is highly conserved among apicomplexans. MORN1 specifically localizes to ring structures at the apical and posterior end of the inner membrane complex and to the centrocone, a specialized nuclear structure that organizes the mitotic spindle. Time-lapse imaging of tagged MORN1 revealed that these structures are highly dynamic and appear to play a role in nuclear division and daughter cell budding. Overexpression of MORN1 resulted in severe but specific defects in nuclear segregation and daughter cell formation. We hypothesize that MORN1 functions as a linker protein between certain membrane regions and the parasite's cytoskeleton. Our initial biochemical analysis is consistent with this model. Whereas recombinant MORN1 produced in bacteria is soluble, in the parasite MORN1 was associated with the cytoskeleton after detergent extraction. (+info)
Effects of de- and rehydration on food-conducting cells in the moss Polytrichum formosum: a cytological study.
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BACKGROUND AND AIMS: Moss food-conducting cells (leptoids and specialized parenchyma cells) have a highly distinctive cytology characterized by a polarized cytoplasmic organization and longitudinal alignment of plastids, mitochondria, endoplasmic reticulum and vesicles along endoplasmic microtubules. Previous studies on the desiccation biology of mosses have focused almost exclusively on photosynthetic tissues; the effects of desiccation on food-conducting cells are unknown. Reported here is a cytological study of the effects of de- and rehydration on food-conducting cells in the desiccation-tolerant moss Polytrichum formosum aimed at exploring whether the remarkable subcellular organization of these cells is related to the ability of mosses to survive desiccation. METHODS: Shoots of Polytrichum formosum were dehydrated under natural conditions and prepared for transmission and scanning electron microscopy using both standard and anhydrous chemical fixation protocols. Replicate samples were then fixed at intervals over a 24-h period following rehydration in either water or in a 10 microM solution of the microtubule-disrupting drug oryzalin. KEY RESULTS: Desiccation causes dramatic changes; the endoplasmic microtubules disappear; the nucleus, mitochondria and plastids become rounded and the longitudinal alignment of the organelles is lost, though cytoplasmic polarity is in part retained. Prominent stacks of endoplasmic reticulum, typical of the hydrated condition, are replaced with membranous tubules arranged at right angles to the main cellular axis. The internal cytoplasm becomes filled with small vacuoles and the plasmalemma forms labyrinthine tubular extensions outlining newly deposited ingrowths of cell wall material. Whereas plasmodesmata in meristematic cells at the shoot apex and in stem parenchyma cells appear to be unaffected by dehydration, those in leptoids become plugged with electron-opaque material. Starch deposits in parenchyma cells adjoining leptoids are depleted in desiccated plants. Rehydration sees complete reestablishment over a 12- to 24-h period of the cytology seen in the control plants. Oryzalin effectively prevents leptoid recovery. CONCLUSIONS: The results point to a key role of the microtubular cytoskeleton in the rapid re-establishment of the elaborate cytoplasmic architecture of leptoids during rehydration. The reassembly of the endoplasmic microtubule system appears to dictate the time frame for the recovery process. The failure of leptoids to recover normal cytology in the presence of oryzalin further underlines the key role of the microtubules in the control of leptoid cytological organization. (+info)
Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening.
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Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening. (+info)
Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment.
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The tri-iodide-based chemiluminescence assay is the most widely used methodology for the detection of S-nitrosothiols (RSNOs) in biological samples. Because of the low RSNO levels detected in a number of biological compartments using this assay, criticism has been raised that this method underestimates the true values in biological samples. This claim is based on the beliefs that (i) acidified sulfanilamide pretreatment, required to remove nitrite, leads to RSNO degradation and (ii) that there is auto-capture of released NO by heme in the reaction vessel. Because our laboratories have used this assay extensively without ever encountering evidence that corroborated these claims, we sought to experimentally address these issues using several independent techniques. We find that RSNOs of glutathione, cysteine, albumin, and hemoglobin are stable in acidified sulfanilamide as determined by the tri-iodide method, copper/cysteine assay, Griess-Saville assay and spectrophotometric analysis. Quantitatively there was no difference in S-nitroso-hemoglobin (SNOHb) or S-nitroso-albumin (SNOAlb) using the tri-iodide method and a recently described modified assay using a ferricyanide-enhanced reaction mix at biologically relevant NO:heme ratios. Levels of SNOHb detected in human blood ranged from 20-100 nM with no arterial-venous gradient. We further find that 90% of the total NO-related signal in blood is caused by erythrocytic nitrite, which may partly be bound to hemoglobin. We conclude that all claims made thus far that the tri-iodide assay underestimates RSNO levels are unsubstantiated and that this assay remains the "gold standard" for sensitive and specific measurement of RSNOs in biological matrices. (+info)