Structural changes of spores of tree fungal pathogens after treatment with the designed antimicrobial peptide D2A21

2000 ◽  
Vol 78 (4) ◽  
pp. 462-471 ◽  
Author(s):  
D Rioux ◽  
V Jacobi ◽  
M Simard ◽  
R C Hamelin
Keyword(s):  
Plasma Membrane ◽  
Spore Germination ◽  
Fungal Pathogens ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Cronartium Ribicola ◽  
Ophiostoma Ulmi ◽  
Nectria Galligena ◽  
In Vitro Effects

In vitro effects of the antimicrobial synthetic D2A21 peptide on the structure of spores of four fungal pathogens causing important tree diseases were examined by microscopy in parallel with tests to measure inhibition of spore germination. With light microscopy, the use of SYTOX®green stain indicated that the peptide rapidly altered the plasma membrane of conidia of three Ascomycetes: Gremmeniella abietina (Lagerberg) Morelet var. abietina Petrini et al., Ophiostoma ulmi (Buism.) Nannf., and Nectria galligena Bres. With basidiospores of Cronartium ribicola J.C. Fisch., a difference between control and treated spores was also found, but it was less pronounced than with conidia of the Ascomycetes. In transmission electron microscopy, untreated conidia showed typical cytoplasmic contents with the regular presence of mitochondria, ribosomes, and nuclei, at times accompanied by vacuoles of various sizes. At concentrations of the peptide inhibitory to spore germination, plasma membranes, as well as nuclear and mitochondrial envelopes, were either generally difficult to discern or were distorted and swollen. At more advanced stages, the cytoplasm of treated spores contained numerous vesicles and was in places more electron-dense than in controls. Cytoplasm leakage was also regularly observed. Present observations strongly suggest that the primary site of action of this peptide is located at the plasma membrane level.Key words: Cronartium ribicola, Gremmeniella abietina, Nectria galligena, Ophiostoma ulmi, D2A21, peptide.

scholarly journals Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 583-594 ◽  
Author(s):  
N Dainiak ◽  
CM Cohen
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Mononuclear Cells ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Target Cells ◽  
Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

scholarly journals Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro.

1976 ◽  
Vol 71 (1) ◽  
pp. 136-158 ◽  
Author(s):  
L A Staehelin
Keyword(s):  
Plasma Membranes ◽  
Structural Changes ◽  
Freeze Fracture ◽  
Chloroplast Membranes ◽  
Ps Ii ◽  
Membrane Particles ◽  
Intramembranous Particles ◽  
Membrane Surfaces ◽  
Ps Ii Activity

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.

scholarly journals The Vitamin D Receptor Is Present in Caveolae-Enriched Plasma Membranes and Binds 1α,25(OH)2-Vitamin D3in Vivo and in Vitro

2004 ◽  
Vol 18 (11) ◽  
pp. 2660-2671 ◽  
Author(s):  
Johanna A. Huhtakangas ◽  
Christopher J. Olivera ◽  
June E. Bishop ◽  
Laura P. Zanello ◽  
Anthony W. Norman
Keyword(s):  
Vitamin D ◽  
Plasma Membrane ◽  
Vitamin D Receptor ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Kidney Tissue ◽  
Mouse Lung ◽  
Nuclear Fraction

Abstract The steroid hormone 1α,25(OH)2-vitamin D3 (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). This study characterized the VDRmem present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [3H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D KD binding dissociation constant = 1–3 nm. Our data collectively support the classical VDR being the VDRmem in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [3H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r2 = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [3H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [3H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

scholarly journals Phospholipase C activation by ethanol in rat hepatocytes is unaffected by chronic ethanol feeding

1990 ◽  
Vol 272 (1) ◽  
pp. 59-64 ◽  
Author(s):  
J B Hoek ◽  
T F Taraschi ◽  
K Higashi ◽  
E Rubin ◽  
A P Thomas
Keyword(s):  
Phospholipase C ◽  
Order Parameter ◽  
Plasma Membranes ◽  
Structural Changes ◽  
Rat Hepatocytes ◽  
Maximal Response ◽  
Molecular Order ◽  
Transient Activation ◽  
Ethanol Feeding

The activation of phosphoinositide-specific phospholipase C by ethanol was compared in hepatocytes isolated from ethanol-fed rats and from pair-fed control animals. Ethanol (100-300 mM) caused a dose-dependent transient increase in cytosolic free Ca2+ levels in indo-1-loaded hepatocytes from both groups of animals. The rate of Ca2+ increase was similar in hepatocytes from control and ethanol-fed rats, but the decay of the Ca2+ increase was somewhat slower in the latter preparation. The ethanol-induced Ca2+ increase caused activation of glycogen phosphorylase, with 50% response at 50 mM-ethanol and a maximal response at 150-200 mM-ethanol, not significantly different in hepatocytes from control and ethanol-fed animals. Ins(1,4,5)P3 formation in response to ethanol (300 mM) or vasopressin (2 nM or 40 nM) was also similar in the two preparations. It is concluded that long-term ethanol feeding does not lead to an adaptive response with respect to the ethanol-induced phospholipase C activation in rat hepatocytes. The ability of ethanol in vitro to decrease membrane molecular order in liver plasma membranes from ethanol-fed and control rats was measured by e.s.r. Membranes from ethanol-fed animals had a significantly lower baseline order parameter compared with control preparations (0.313 and 0.327 respectively), indicative of decreased membrane molecular order. Addition of 100 mM-ethanol significantly decreased the order parameter in control preparations by 2.1%, but had no effect on the order parameter of plasma membranes from ethanol-fed rats, indicating that the plasma membranes had developed tolerance to ethanol, similar to other membranes in the liver. Thus the membrane structural changes associated with this membrane tolerance do not modify the ethanol-induced activation of phospholipase C. The transient activation of phospholipase C by ethanol in hepatocytes may play a role in maintaining an adaptive phenotype in rat liver.

scholarly journals In vitro maintenance of boar sperm viability by a soluble fraction obtained from oviductal apical plasma membrane preparations

Reproduction ◽  
2003 ◽  
pp. 509-517 ◽  
Author(s):  
A Fazeli ◽  
RM Elliott ◽  
AE Duncan ◽  
A Moore ◽  
PF Watson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Current Knowledge ◽  
Biologically Active ◽  
Apical Plasma Membrane ◽  
Sperm Viability ◽  
Boar Sperm ◽  
Membrane Contact ◽  
Vitro Model

Oviductal apical plasma membrane fractions have been successfully used to provide an in vitro model to study the role of direct membrane contact in sperm-oviduct interactions. Apical plasma membrane preparations from pig oviductal tissues show a dose-response in their ability to maintain boar sperm viability in vitro. Membrane preparations obtained from other tissues (lung and duodenum) are incapable of maintaining boar sperm viability to the same extent as oviductal tissue. The present study examined the validity of two hypotheses that arise from current knowledge of sperm-oviduct interactions, namely, that (i) apical plasma membranes prepared from ampullar regions of the oviduct are less effective than those from isthmus regions, and (ii) sperm survival is more effective in apical plasma membrane preparations derived from follicular phase oviducts than those derived from luteal phase oviducts. Both hypotheses were proved false. The nature of the active component(s) in the oviductal apical plasma membrane fractions was further investigated. Heat treatment (100 degrees C for 20 min) diminished the capacity of membranes to support boar sperm viability. Furthermore, a soluble salt-extracted fraction obtained from oviductal apical plasma membrane preparations was biologically active and supported boar sperm viability in vitro. This may indicate that the active factor(s) responsible for the maintenance of boar sperm viability is not an integral part of oviductal membranes and is peripherally bound to these membranes.

scholarly journals Nanosilver Colloid Inhibits Toxoplasma gondii Tachyzoites and Bradyzoites in Vitro

2019 ◽  
Author(s):  
Saeedeh SHOJAEE ◽  
Nima FIROUZEH ◽  
Hussein KESHAVARZ ◽  
Sanaz JAFARPOUR AZAMI ◽  
Mahboobeh SALIMI ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Structural Changes ◽  
Protozoan Parasite ◽  
Blue Dye ◽  
Type I ◽  
Worldwide Distribution ◽  
Life Threatening ◽  
Strain Type ◽  
In Vitro Effects

Background: Toxoplasma gondii, the coccidian protozoan parasite with worldwide distribution, is the agent of toxoplasmosis. The disease is life threatening in congenital form and in immunocompromised patients. The present study was carried out in 2016 to evaluate the in vitro effects of nanosilver colloid on tachyzoites and bradyzoites of T. gondii, RH and Tehran strains. Methods: Different concentrations (5, 10 , 20 ppm) of  nanosilver colloid were added to tachyzoites of T. gondii , RH strain (type I) and bradyzoites and tissue cysts of T. gondii , Tehran strain (type II) and incubated for 30, 60, 90 and 120 minutes. The mortality rates of tachyzoites and bradyzoites were evaluated by trypan blue dye and MTT assay. Then SEM carried out to show the changes between control and exposed parasites. Results: The greatest mortality rate was seen in 20 ppm concentration and after 120 minutes of exposure. By electron microscopy, the structural changes were seen in tachyzoites of RH and tissue cyst of Tehran strain in comparison with control groups. Conclusion: Nanosilver colloid was effective on both tachyzoites and bradyzoites of T. gondii, RH and Tehran strains.

scholarly journals In Vitro Effects of Silicon on the Action Patterns of Sugarcane Acid Invertase

1969 ◽  
Vol 52 (4) ◽  
pp. 311-322
Author(s):  
Alex G. Alexander
Keyword(s):  
Silicic Acid ◽  
Structural Changes ◽  
Gel Filtration ◽  
Acid Invertase ◽  
Sucrose Hydrolysis ◽  
In Vitro Effects ◽  
Acid Gel ◽  
Living Tissues ◽  
Hydrolysis Of

In vitro studies of invertase action patterns were conducted in the presence of silicon (Si). This element is known to inhibit the enzyme both in living tissues and cell-free preparations. Substrates included sucrose, raffinose, stachyose, and turanose. Sugarcane acid invertase was prepared from lyophilized immature storage tissue and was partially purified by salt fractionation, dialysis, and gel filtration. Enzyme products were studied by paper chromatography. Two products, fructose and glucose, were quickly obtained in large quantity from sucrose. Against raffinose the reaction proceeded more slowly but yielded a total of four products. This suggests a powerful work potential of cane invertase since complete acid hydrolysis of raffinose gives only three products. It is proposed that both 1 —> 6 and 1 —> 2 linkages are broken. Fructose appears by direct hydrolysis of raffinose and by secondary attacks upon the intermediate product sucrose. Use of stachyose as substrate gave additional evidence for hydrolysis of the 1 —> 6 linkage. Three products were obtained in the presence of only one 1 —> 2 fructosidic linkage. Incorporation of Si into invertase digests abruptly terminated the reactions against sucrose, raffinose and stachyose. The effective Si concentration was slightly more than 3 µmoles of Si per milliliter of digest. Significance of the invertase inhibition is mentioned both from the standpoint of increasing sucrose yield and as an analytical indicator of silicic acid content of plant tissues. When the disaccharide turanose was employed as substrate, increasing Si levels gave additional products rather than suspended enyme action. Four products appeared in the presence of 9 µmoles of Si. The substrates sucrose and raffinose yielded masses of variably-staining products at Si levels above 27 µmoles per milliliter. The latter products were of low chromatographic mobility and resembled fragments of hydrolyzed starch. To account for Si action on invertase it is proposed that a silicic acid gel forms around the enzyme in direct proportion to Si concentration. Inhibition of sucrose hydrolysis does not stem from severe protein structural changes. Rather, the hypothetical, gel-encased enzyme might cease to function against one substrate while retaining or increasing its capacity to attack others.

Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action

1992 ◽  
Vol 263 (5) ◽  
pp. E974-E979 ◽  
Author(s):  
M. Wehling ◽  
M. Christ ◽  
K. Theisen
Keyword(s):  
Plasma Membranes ◽  
Membrane Receptors ◽  
Mononuclear Leukocytes ◽  
High Turnover ◽  
Apparent Dissociation Constant ◽  
In Vitro Effects ◽  
Nongenomic Effects ◽  
Human Mononuclear Leukocytes ◽  
Dissociation Constant Kd

Rapid nongenomic in vitro effects of aldosterone on intracellular electrolytes, cell volume, and Na(+)-H+ antiport have been found in human mononuclear leukocytes (HML). Binding of 125I-labeled aldosterone to plasma membranes of HML shares important features with these functional data. This includes a very low apparent dissociation constant (Kd) of 0.1 nM for both aldosterone and the effect on the Na(+)-H(+)-antiport, a high turnover rate, and the almost exclusive binding selectivity for aldosterone. Dexamethasone, RU 26988, corticosterone, ouabain, amiloride, and 18-hydroxyprogesterone were inactive as ligands. Deoxycorticosterone acetate had an intermediate activity with an apparent Kd of 100 nM. These findings are the first to demonstrate membrane binding of aldosterone being compatible with major aspects of its nongenomic effects.

Mechanisms responsible for SCN increase in resistance of in vitro frog gastric mucosa

1983 ◽  
Vol 245 (1) ◽  
pp. G143-G156 ◽  
Author(s):  
W. S. Rehm ◽  
T. C. Chu ◽  
M. Schwartz ◽  
G. Carrasquer
Keyword(s):  
Plasma Membrane ◽  
Gastric Mucosa ◽  
Plasma Membranes ◽  
Concentration Profiles ◽  
Tubular Cells ◽  
Nacl Concentration ◽  
Separate Site ◽  
Hcl Secretion ◽  
High Conductance

Thiocyanate (SCN) inhibits H+ secretion and increases the resistance and potential difference (PD) of the gastric mucosa. These results support our separate-site electrogenic theory of HCl secretion. Recent work shows that an ATP-driven mechanism in the gastric mucosa can produce H+ by a neutral exchange of K+ for H+. The SCN increase in resistance and PD, if due to an inhibition of a high-conductance mechanism(s) in the secretory plasma membrane, is not easily compatible with the neutral mechanism. Therefore, the possibility was examined that SCN induces the increase in resistance by other mechanisms. The HCl and NaCl concentration profiles in the pit and tubular lumina were calculated. The effects of SCN were determined with isotonic, hypotonic, hypertonic, buffered, and high [H+] secretory solutions. The results indicate that SCN produces an increase in resistance of about 130 omega. cm2 of the plasma membranes of the tubular cells. A scheme is proposed that incorporates the neutral K+-H+ mechanism into an electrogenic system.

In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots

1992 ◽  
Vol 84 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Marta Roldan ◽  
Andres Belver ◽  
Pilar Rodriguez-Rosales ◽  
Nuria Ferrol ◽  
Juan Pedro Donaire
Keyword(s):  
Plasma Membrane ◽  
Proton Pump ◽  
In Vitro Effects ◽  
Plasma Membrane Proton Pump
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