scholarly journals Dissection of the Ascaris Sperm Motility Machinery Identifies Key Proteins Involved in Major Sperm Protein-based Amoeboid Locomotion

2003 ◽  
Vol 14 (12) ◽  
pp. 5082-5088 ◽  
Author(s):  
Shawnna M. Buttery ◽  
Gail C. Ekman ◽  
Margaret Seavy ◽  
Murray Stewart ◽  
Thomas M. Roberts
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Membrane Vesicles ◽  
Major Sperm Protein ◽  
Plasma Membrane Vesicles ◽  
Sperm Protein ◽  
Fiber Assembly ◽  
A Cell ◽  
Fiber Proteins

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.

scholarly journals Hydrostatic Pressure Shows That Lamellipodial Motility in Ascaris Sperm Requires Membrane-associated Major Sperm Protein Filament Nucleation and Elongation

1998 ◽  
Vol 140 (2) ◽  
pp. 367-375 ◽  
Author(s):  
Thomas M. Roberts ◽  
E.D. Salmon ◽  
Murray Stewart
Keyword(s):  
Plasma Membrane ◽  
Hydrostatic Pressure ◽  
Leading Edge ◽  
Major Sperm Protein ◽  
Sperm Protein ◽  
Fiber Assembly ◽  
Fiber Growth ◽  
Filament Assembly

Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in place of an actin cytoskeleton to drive their ameboid locomotion. Motility is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been reconstituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle forward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, and C.A. Fontana. 1996. Cell. 84:105–114). We used changes in hydrostatic pressure within a microscope optical chamber to investigate the mechanism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vitro system to show that filament assembly occurs in the immediate vicinity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, normal fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number of filaments assembled at the vesicle (proportional to fiber optical density and filament nucleation rate), and their rate of assembly (proportional to the rates of fiber growth and filament elongation). Thus, fiber growth is not simply because of the addition of subunits onto the ends of existing filaments, but rather is regulated by pressure-sensitive factors at or near the vesicle surface. Once a filament is incorporated into a fiber, its rates of addition and loss of subunits are very slow and disassembly occurs by pathways distinct from assembly. The effects of pressure on fiber assembly are sensitive to dilution of the extract but largely independent of MSP concentration, indicating that a cytosolic component other than MSP is required for vesicle-association filament nucleation and elongation. Based on these data we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading edge.

Alanine uptake by liver at midpregnancy in rats

1987 ◽  
Vol 252 (3) ◽  
pp. E408-E413 ◽  
Author(s):  
M. Pastor-Anglada ◽  
X. Remesar ◽  
G. Bourdel
Keyword(s):  
Plasma Membrane ◽  
Active Transport ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Pregnant Rats ◽  
Dependent Transport

The participation of the liver to the increase in alanine utilization seen at midpregnancy was studied in 9- and 12-day pregnant rats. Liver fractional extraction of alanine was assessed in vivo from the changes in concentration in afferent and efferent vessels. Hepatic active transport of alanine was determined in vitro using isolated plasma-membrane vesicles. Compared with nonpregnant controls, alanine fractional extraction was significantly increased on day 12 but not on day 9 of pregnancy. Vesicles isolated from 9- and 12-day pregnant animals had a greater capacity for Na+-dependent transport than those from controls. Eadie-Hofstee plotting showed that this increase was due to an increase in Vmax with no change in Km. Both A and ASC systems contributed to the Vmax increase. These results indicate that, although by day 9 the liver has developed an increased capacity for alanine uptake, the actual extraction is seen only by day 12 of pregnancy. At this stage the liver participates actively in the turnover of alanine and the development of hypoalaninemia.

scholarly journals 131 PEROXIDATIVE CHALLENGE OF PLASMA MEMBRANE VESICLES FROM BELL PEPPER FRUIT LEADS TO DECREASED H+ATPase ACTIVITY

HortScience ◽  
1994 ◽  
Vol 29 (5) ◽  
pp. 447c-447
Author(s):  
Darlene M. Cowart ◽  
Robert L. Shewfelt
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Fruits And Vegetables ◽  
Chilling Injury ◽  
Membrane Vesicles ◽  
Thiobarbituric Acid ◽  
Plasma Membrane Vesicles ◽  
Water Control ◽  
Solubilized Enzyme

-Lipid peroxidation has been proposed as an important factor in chilling injury of susceptible fruits and vegetables. The effect of in vitro peroxidative challenge on H+ATPase activity in intact plasma membrane vesicles and solubilized enzyme was determined by incubation with (1) deionized water (control), (2) Fe3+-ascorbate, and (3) lipoxygenase (LOX) + phospholipase A2(PLA2) for 0, 30, and 60 min. Enzyme activity increased throughout the incubation period with no accumulation of thiobarbituric acid-reactive substances (TBA-RS) in the control, but vesicles challenged by the peroxidative systems showed significant increases in TBA-RS and decreases in membrane-bound H+ATPase activity. Greater losses in H+ATPase activity were observed in solubilized enzyme than in intact vesicles. The results indicate that loss of H+ATPase activity due to chemical modification of the protein rather than changes in membrane fluidity and suggest that modification is away from the active site.

Differences in L-alanine uptake by livers of Wistar and lean Zucker rats

1991 ◽  
Vol 11 (2) ◽  
pp. 85-93
Author(s):  
B. Ruiz ◽  
J. Casado ◽  
M. Pastor-Anglada ◽  
A. Felipe
Keyword(s):  
Plasma Membrane ◽  
Wistar Rats ◽  
Membrane Vesicles ◽  
Hepatic Uptake ◽  
Zucker Rats ◽  
Plasma Membrane Vesicles ◽  
Blood Flows ◽  
Old Rats ◽  
Lower Capacity

The L-alanine uptake by livers of Wistar and lean Zucker rats has been studied. The hepatic uptake and fractional extraction rates of alanine were estimated in 50–55 day old rats. No significant differences in amino acid concentrations and blood flows in afferent and efferent liver vessels were seen in lean Zucker rats when compared with Wistar rats. However, the hepatic uptake (1.6±0.1 and 0.7±0.1 μmol/min/100 g bw, p<0.01) and the fractional extraction (26.8±2.1 and 15.2±3.1%, p<0.05) were much lower in Zucker than in Wistar rats. The hepatic active transport of L-alanine was determined in vitro using isolated plasma membrane vesicles. Vesicles isolated from livers of lean Zucker rats showed similar values of Km (2.5±0.7 vs 2.0±0.5 mM for Wistar and Zucker respectively, N.S.), but lower values of Vmax when compared with Wistar rats (1.1±0.1 vs 0.6±0.005 nmol/mg prot 5 s, p<0.01, for Wistar and lean Zucker rats respectively). These results indicate that, the liver of lean Zucker rats concentrates alanine less efficiently than the liver of Wistar rats. This fact correlates well with a lower capacity of the Na+-dependent L-alanine trasport in liver plasma membrane vesicles from lean Zucker rats.

scholarly journals Delayed restoration of Mg2+ content and transport in liver cells following ethanol withdrawal

2009 ◽  
Vol 297 (4) ◽  
pp. G621-G631 ◽  
Author(s):  
Lisa M. Torres ◽  
Christie Cefaratti ◽  
Liliana Berti-Mattera ◽  
Andrea Romani
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Ethanol Withdrawal ◽  
Adrenergic Stimulation ◽  
Plasma Membrane Vesicles ◽  
Isolated Cells ◽  
Intact Cells

Liver cells from rats chronically fed a Lieber-De Carli diet for 3 wk presented a marked decreased in tissue Mg2+ content and an inability to extrude Mg2+ into the extracellular compartment upon stimulation with catecholamine, isoproterenol, or cell-permeant cAMP analogs. This defect in Mg2+ extrusion was observed in both intact cells and purified liver plasma membrane vesicles. Inhibition of adrenergic or cAMP-mediated Mg2+ extrusion was also observed in freshly isolated hepatocytes from control rats incubated acutely in vitro with varying doses of ethanol (EtOH) for 8 min. In this model, however, the defect in Mg2+ extrusion was observed in intact cells but not in plasma membrane vesicles. In the chronic model, upon removal of EtOH from the diet hepatic Mg2+ content and extrusion required ∼10 days to return to normal level both in isolated cells and plasma membrane vesicles. In hepatocytes acutely treated with EtOH for 8 min, more than 60 min were necessary for Mg2+ content and extrusion to recover and return to the level observed in EtOH-untreated cells. Taken together, these data suggest that in the acute model the defect in Mg2+ extrusion is the result of a limited refilling of the cellular compartment(s) from which Mg2+ is mobilized upon adrenergic stimulation rather than a mere defect in adrenergic cellular signaling. The chronic EtOH model, instead, presents a transient but selective defect of the Mg2+ extrusion mechanisms in addition to the limited refilling of the cellular compartments.

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459 ◽  
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

Abstract In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

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