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At least 73 records · Page 4Linked to original sources
PubMed1988

Kinetics of fluorescent-labeled phosphatidylcholine transfer between nonspecific lipid transfer protein and phospholipid vesicles.

Recently, rat liver nonspecific lipid transfer protein (nsLTP) was shown to form a fluorescent complex when allowed to equilibrate with self-quenching vesicles prepared from the fluorescent phospholipid 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (P-C12-NBD-PC) [Nichols, J. W. (1987) J. Biol. Chem. 262, 14172-14177]. Investigation of the mechanism of complex formation was continued by studying the kinetics of transfer of P-C12-NBD-PC between nsLTP and phospholipid vesicles using a transfer assay based on resonance energy transfer between P-C12-NBD-PC and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine. The principles of mass action kinetics (which predict initial lipid transfer rates as a function of protein and vesicle concentration) were used to derive equations for two distinct mechanisms: lipid transfer by the diffusion of monomers through the aqueous phase and lipid transfer during nsLTP-membrane collisions. The results of these kinetics studies indicated that the model for neither mechanism alone adequately predicted the initial rates of formation and dissolution of the P-C12-NBD-PC-nsLTP complex. The initial rate kinetics for both processes were predicted best by a model in which monomer diffusion and collision-dependent transfer occur simultaneously. These data support the hypothesis that the phospholipid-nsLTP complex functions as an intermediate in the transfer of phospholipids between membranes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1985

Transfer of label from 3H-glucose in Digitaria eriantha leaves to the rust fungus Puccinia digitariae Pole Evans.

Digitaria eriantha pentzii was fed 3H-glucose prior to inoculation with uredospores of Puccinia digitariae Pole Evans. Twenty-one hours after inoculation, uptake of label from 3H-glucose by the primary infection structures of P. digitariae was demonstrated employing autoradiography. These results indicate that an exchange of nutrients between host and pathogen occurs very early on in the infection process, during the formation of the primary infection structures. Despite contrary reports that obligate parasites receive no nutrition before establishment of haustoria, this study supports the work of Andrews (Can J Bot 53:1103, 1975), who demonstrated uptake of 3H-glucose label from lettuce cotyledons into the primary and secondary infection vesicles, appressoria, and germ tubes of Bremia lactucae.

Autoradiography↗
PubMed1968

The dual effect of lithium ions on sodium efflux in skeletal muscle.

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3 degrees C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer's solution containing 60 mM LiCl and 50 mM NaCl at 20 degrees C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10(-5)M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer's solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10(-5)M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer's solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.

Animals↗
PubMed1994

Host leukocytes and their products in chronic kidney allograft rejection in rats.

Bilaterally nephrectomized Lewis recipients of Fisher 344 (F344) kidney allografts, treated with CyA (1.5 mg/kg/day x 10), develop progressive changes of chronic rejection. Treated F344-to-F344 acted as isograft controls. Proteinuria was determined sequentially. Grafts were harvested 8, 12 and 16 weeks after transplantation (n = 9/group/time period). Infiltrating host cells and their products were assessed in chronically rejecting grafts by histology and immunostaining using mAbs for monocyte/macrophages, T-cells, ICAM-1, LFA-1 and cytokines. For in vitro binding studies, snap-frozen sections of transplanted kidneys were incubated with monocytes/macrophages and lymphocytes isolated from peripheral blood (PBL) of naive animals. For in vivo migration studies, naive cell populations were labeled with Bis-Benzamide and transferred i.v. to grafted animals at weeks 8, 12 and 16 (n = 3/group); grafts were harvested 24 h later and cell localization assessed under immunofluorescence. Increasing numbers of ED1 + monocytes/macrophages in allografted kidneys peaked at 16 weeks, localizing preferentially in glomeruli, where IL-1, IL-6 and TNF-alpha expression had also become intense and correlated with progressive glomerulosclerosis. Binding studies corroborated these results. In vitro, a few monocytes/macrophages bound to glomeruli and vessels at 8 weeks; by 12 weeks, binding to glomeruli was high (72% of cells). In vivo, large numbers of transferred labelled monocytes/macrophages were found in kidney allografts at 12 weeks (23%, isografts; < 7%, P < 0.01). In contrast T cells (primarily CD4+) were a consistent feature in allografts elevated as compared to isografts and correlating with in vitro and in vivo binding patterns; associated cytokines included IL-2, IFN- and TNF-alpha. Functional data followed these results: urine protein excretion by allograft recipients increased from baseline at 8 weeks (12 mg/day) to > 50 mg/day at 16 weeks at which point animals were beginning to die of renal failure; proteinuria in isografted rats did not increase during this time period. These results suggest that monocyte/macrophage and CD4+ T cells and their products are important in chronic kidney allograft rejection, contributing to the progressive sclerosis and fibrosis.

Animals↗
PubMed1990

Rapid detection of proteins by enzyme-linked immunofiltration assay after transfer onto nitrocellulose membranes.

Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 micron). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetitive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.

Animals↗
PubMed1989

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.

Acetates↗
PubMed1991

Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver.

Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.

Adenosine Triphosphate↗
PubMed2004

Multiple-spin analysis of chemical-shift-selective (13C, 13C) transfer in uniformly labeled biomolecules.

Chemical-shift-selective (13C, 13C) polarization transfer is analyzed in uniformly labeled biomolecules. It is shown that the spin system dynamics remain sensitive to the distance of interest and can be well reproduced within a quantum-mechanical multiple-spin analysis. These results lead to a general approach on how to describe chemical-shift-selective transfer in uniformly labeled systems. As demonstrated in the case of ubiquitin, this methodology can be used to detect long-range distance constraints in uniformly labeled proteins.

Biopolymers↗
PubMed1983

The relationship between placental protein synthesis and transfer of amino acids.

The relationship between placental protein synthesis and transfer of amino acids from mother to foetus was studied in the guinea pig, by using [U-14C]-lysine, -leucine, -glycine, -aspartate and -alpha-aminoisobutyrate. The uptake of label by protein was 12-16% of total label transferred. Cycloheximide inhibited incorporation of all naturally occurring amino acids into protein by 81-96% and transfer by 62-75%; the concentration of label in the free pool was increased for each. These findings were confirmed when specific-radioactivity measurements were made with L-[U-14C]lysine. The transfer of the non-protein amino acid alpha-aminoisobutyrate was not significantly decreased by cycloheximide. A model, linking protein synthesis to the generation of a transfer pool of amino acids, is proposed whereby inhibition of protein synthesis decreases the amount of amino acid available for transfer.

Amino Acids↗
PubMedDate not supplied

D-glucose transfer rates derived from single-injection and steady state tracer experiments in the isolated guinea-pig placenta.

In isolated guinea-pig placentae the transfer of D-[3H]glucose and L-[14C]glucose was investigated in single injection (SIE) and steady state (SSE, n = 11, placental weight 4.5 +/- 1.14 g) experiments at constant perfusion flow rates (3 ml/min). In SIE, a mixture of D- and L-glucose was injected as a bolus into either the fetal or maternal side of the placenta, uptake curves were obtained and the maximal extraction values Umax were derived. From these the membrane rate constants Kmc and Kfc of either the maternal or fetal side of the trophoblast membrane were calculated. The specific placental transfer rate constants were computed for the maternal-fetal (Kspec,mf) and fetal-maternal (Kspec,fm) direction from the amount of label transferred to the acceptor side in either SIE or in SSE (where labelled D- and L-glucose had been added to the stock solution). The chemical concentration of D-glucose was changed (5, 50 and 100 mmol/l, n = 11) and it was found that all rate constants decreased with increasing D-glucose concentration. At a D-glucose concentration of 5 mmol/l, Kmc was 3.11 +/- 1.59 ml/min (n = 6) and Kfc 2.69 +/- 0.5 ml/min (n = 5), the combined membrane rate constant (which determines the total placental transfer) was estimated to be 1.44 ml/min. This value was not significantly smaller than the mean (both directions) transfer rate constant Kspec in SIE (1.51 +/- 0.89 ml/min) or SSE (1.52 +/- 0.56 ml/min). Thus the results from uptake and transfer experiments are consistent. From the rate constants D-glucose fluxes at different chemical D-glucose concentrations were estimated. Whereas the mean Km values for all fluxes based on the various rate constants were about 30 mmol/l, the maximal flux Vmax was highest at the maternal trophoblast membrane (159.1 +/- 70.2 mumol/min), it was 107.9 +/- 11.6 mumol/min at the fetal side and 67.3 +/- 7.9 mumol/min in SIE or SSE for placental transfer in both directions. It is concluded that D-glucose carriers predominate at the maternal side of the trophoblast, and that maternal-fetal glucose transfer is proportionate to the transplacental glucose concentration difference within the physiological range of glucose concentrations.

Animals↗