Translational diffusion of individual class II MHC membrane proteins in cells

Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells.     

Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane

Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.     

Mouse B lymphocyte specific endocytosis and recycling of MHC class II molecules

In B lymphocytes, the processing of exogenous proteins and the subsequent binding of antigenic peptides to class II molecules encoded by the major histocompatibility complex (MHC) occurs most likely within endocytic compartments. To examine the endocytic transport of MHC class II molecules, we used (i) surface iodination followed by internalization, pronase treatment and immunoprecipitation, (ii) in situ iodination of endosomal compartments, and (iii) confocal microscopy to visualize the fate of fluorescence coupled Fab fragments. In murine I-Ak, I-Ek positive B lymphoma cells, cell surface MHC class II molecules are partially protected from pronase digestion after 15 min at 37 degrees C and recycled back to the cell surface within the next 30 min. The fluorescence coupled Fab fragments are delivered to juxtanuclear endocytic compartments in 15 min. In contrast to the murine B cells, L fibroblasts transfected with either I-A alpha beta k or I-E alpha k beta k,d fail to internalize their surface class II molecules. A fraction of class II molecules, however, is still present in endosomal compartments as detected after in situ iodination in L fibroblasts. We conclude that the recipient L fibroblasts lack one or several factors needed for the transport of MHC class II molecules from the cell surface to the endosomes. We suggest that in murine B lymphoma cells, antigenic peptides can gain access to a pool of recycling class II molecules whereas in L cells they meet newly synthesized class II molecules targeted to the endosomal compartments.     

Contribution of a single hydrogen bond between betaHis81 of MHC class II I-E(k) and the bound peptide to the pH-dependent thermal stability

To determine the energetic contribution of the hydrogen bond between betaHis81 of the major histocompatibility complex class II (MHC II) molecule, I-E(k), and the bound hemoglobin peptide (Hb), we analyzed the thermal stability of the hydrogen bond-disrupted mutant, I-E(k)-Hb betaH81Y, in which the betaHis81 residue was replaced with Tyr, by differential scanning calorimetry. The thermal stability of the I-E(k)-Hb betaH81Y mutant was lower than that of the I-E(k)-Hb wild-type, mainly due to the decreased enthalpy change. The difference in the denaturation temperature of the I-E(k)-Hb betaH81Y mutant as compared with that of the I-E(k)-Hb wild-type at pH 5.5 was only slightly smaller than that at pH 7.4, in agreement with the increased stability at an acidic pH, a unique characteristic of MHC II. Thus, the hydrogen bond contributed by betaHis81 is critical for peptide binding, and is independent of pH, which can alter the hydrophilicity of the His residue.     

Kinetics of registry selection of chimeric peptides binding to MHC II

Major histocompatability complex type II proteins (MHC II) are alphabeta-heterodimeric glycoproteins that present peptides to the T cell receptor (TCR) of CD4(+) T-cells. This presentation may result in activation of these T-cells, depending on the nature of the peptide. Peptides interact specifically with MHC II with nine peptide amino acid positions, and the corresponding MHC II pocket positions are usually labeled P1-P9. However, the length of peptides binding to MHC II may be greater than nine amino acids, and therefore these peptides may potentially bind to the MHC II in more than one registry. To investigate the mechanism by which a long peptide binds to I-E(k), a murine MHC II, a chimeric peptide with two nonoverlapping registries, f-IAYLKQATKQLRMATPLLMR was designed. The IAYLKQATK peptide segment is based on moth cytochrome c 95-103 (MCC 95-103), and the QLRMATPLLMR segment is based on murine Ii CLIP 89-99 M90L (Ii CLIP 89-99 M90L). This chimeric peptide forms two isomeric complexes. The MCC and Ii CLIP registries dissociate from I-E(k) with t(1/2) values of >800 and 4.94 h, respectively. The registry composition of this MHC II/chimeric peptide complex was found to change as a function of time in approaching thermodynamic equilibrium: the results are consistent with a kinetic model that involves no intramolecular isomer interconversion. The model depicts uncorrelated binding to the MHC II determined by relative association rates to the two registries. This is followed by dissociation and subsequent rebinding, leading ultimately to a preponderance of the most stable complex. Similar results were obtained at pH 5.3. The behavior of this chimeric peptide approximates the binding of a 1:1 solution mixture of two peptides to MHC II, where the more stable complex is selected over time. We have also found that a chimeric peptide and a human MHC II, HLA-DR40401, form isomers with relative association rates to DR0401 at pH 5.3 of 15% for one isomer and 85% for the second isomer.     

Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface

Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta- GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta- GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway.     

Characterization of norepinephrine binding to plasma proteins of the dog and the rabbit

The binding of 3H-norepinephrine (L-3H-NE, 1.0 X 10(-9) M) to plasma proteins of the dog and the rabbit was studied under controlled conditions. Destruction of NE occurred less rapidly at 22 degrees than at 37 degrees. Binding measured at 22 degrees was equivalent to that at 37 degrees, while binding measured at 0 degree was greater than that at 37 degrees. Therefore, losses of plasma NE were minimized by incubation of samples at 22 degrees for no longer than 30 minutes. L-3H-NE binding was examined in the absence and presence of 10(-9) to 10(-2) M non-labeled L-NE, DL-NE, DL-normetanephrine (NM), DL-epinephrine (E), dopamine (DA), and catechol (C). Specific binding of L-3H-NE varied in the range of NE concentrations (L-3H-NE + non-labeled NE) from 10(-9) M (18.7 +/- 3.1%, rabbit; 25.6 +/- 4.8%, dog) to 10(-6) M (10.8 +/- 3.1%, rabbit; 15.2 +/- 3.6%, dog). Calculated binding constants (KD) were consistent with binding to circulating proteins such as globulins or albumin (4.2 +/- 1.2 X 10(-5) M, rabbit; 5.4 +/- 1.7 X 10(-5) M, dog). In plasma from both species, non-labeled DL-NE, L-NE, E, DA, and C, but not NM (from 10(-9) to 10(-2) M) each significantly displaced L-3H-NE from its binding site in a manner similar to displacement produced by non-labeled NE. The results demonstrate that 1) NE is bound to plasma proteins, although to a lesser extent than had been reported by other investigators; and 2) the binding of catecholamines to plasma proteins may be mediated by the catechol ring.     

Invariant chain and the MHC class II cytoplasmic domains regulate localization of MHC class II molecules to lipid rafts in tumor cell-based vaccines

Cell-based tumor vaccines, consisting of MHC class I+ tumor cells engineered to express MHC class II molecules, stimulate tumor-specific CD4+ T cells to mediate rejection of established, poorly immunogenic tumors. Previous experiments have demonstrated that these vaccines induce immunity by functioning as APCs for endogenously synthesized, tumor-encoded Ags. However, coexpression of the MHC class II accessory molecule invariant chain (Ii), or deletion of the MHC class II cytoplasmic domain abrogates vaccine immunogenicity. Recent reports have highlighted the role of lipid microdomains in Ag presentation. To determine whether Ii expression and/or truncation of MHC class II molecules impact vaccine efficacy by altering MHC class II localization to lipid microdomains, we examined the lipid raft affinity of MHC class II molecules in mouse M12.C3 B cell lymphomas and SaI/A(k) sarcoma vaccine cells. Functional MHC class II heterodimers were detected in lipid rafts of both cell types. Interestingly, expression of Ii in M12.C3 cells or SaI/A(k) cells blocked the MHC class II interactions with cell surface lipid rafts. In both cell types, truncation of either the alpha- or beta-chain decreased the affinity of class II molecules for lipid rafts. Simultaneous deletion of both cytoplasmic domains further reduced localization of class II molecules to lipid rafts. Collectively, these data suggest that coexpression of Ii or deletion of the cytoplasmic domains of MHC class II molecules may reduce vaccine efficacy by blocking the constitutive association of MHC class II molecules with plasma membrane lipid rafts.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions

Methyl-beta-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [3H]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.     

Fish oil disrupts MHC class II lateral organization on the B-cell side of the immunological synapse independent of B-T cell adhesion

Fish oil-enriched long chain n-3 polyunsaturated fatty acids disrupt the molecular organization of T-cell proteins in the immunological synapse. The impact of fish oil derived n-3 fatty acids on antigen-presenting cells, particularly at the animal level, is unknown. We previously demonstrated B-cells isolated from mice fed with fish oil-suppressed naïve CD4⁺ T-cell activation. Therefore, here we determined the mechanistic effects of fish oil on murine B-cell major histocompatibility complex (MHC) class II molecular distribution using a combination of total internal reflection fluorescence, Förster resonance energy transfer and confocal imaging. Fish oil had no impact on presynaptic B-cell MHC II clustering. Upon conjugation with transgenic T-cells, fish-oil suppressed MHC II accumulation at the immunological synapse. As a consequence, T-cell protein kinase C theta (PKCθ) recruitment to the synapse was also diminished. The effects were independent of changes in B-T cell adhesion, as measured with microscopy, flow cytometry and static cell adhesion assays with select immune ligands. Given that fish oil can reorganize the membrane by lowering membrane cholesterol levels, we then compared the results with fish oil to cholesterol depletion using methyl-B-cyclodextrin (MβCD). MβCD treatment of B-cells suppressed MHC II and T-cell PKCθ recruitment to the immunological synapse, similar to fish oil. Overall, the results reveal commonality in the mechanism by which fish oil manipulates protein lateral organization of B-cells compared to T-cells. Furthermore, the data establish MHC class II lateral organization on the B-cell side of the immunological synapse as a novel molecular target of fish oil.     

Peptide-specific intercellular transfer of MHC class II to CD4+ T cells directly from the immunological synapse upon cellular dissociation

The transfer of membrane proteins from APC to T cells was initially described in the 1970s, and subsequent work has described two mechanisms of transfer: APC-derived exosomes and direct transfer of small packets, while cells remain conjugated. Using fibroblast APC expressing a GFP-tagged I-E(k) molecule with covalently attached antigenic peptide, we observed a third mechanism in live cell imaging: T cells spontaneously dissociating from APC often capture MHC:peptide complexes directly from the immunological synapse. Using two I-E(k)-restricted murine TCR transgenic T cells with different peptide specificity, we show in this study that the MHC transfer is peptide specific. Using blocking Abs, we found that MHC:peptide transfer in this system requires direct TCR-MHC:peptide interactions and is augmented by costimulation through CD28-CD80 interactions. Capture of the GFP-tagged MHC:peptide complexes correlates with an activated phenotype of the T cell, elevated CD69 with down-modulated TCR. The transferred MHC:peptide molecules transferred to the T cell are associated with molecules that imply continued TCR signaling; p56(lck), phosphotyrosine, and polarization of the actin cytoskeleton.     

Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells

Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4-12 degrees C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 degrees C incubations. At temperatures between 12 and 30 degrees C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 degrees C. Small increases in the extracellular peptide concentration in 37 degrees C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-beta-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.     

Kinetics of cholesterol and phospholipid exchange between Mycoplasma gallisepticum cells and lipid vesicles. Alterations in membrane cholesterol and protein content

The kinetics of exchange of radiolabeled cholesterol and phospholipids between intact Mycoplasma gallisepticum cells and unilamellar lipid vesicles were investigated over a wide range of cholesterol/phospholipid molar ratio. The change in cholesterol/phospholipid molar ratio was achieved by adapting the sterol-requiring M. gallisepticum to grow in cholesterol-poor media, providing cells with decreased unesterified cholesterol content. At least 90% of the cholesterol molecules in unsealed M. gallisepticum membranes underwent exchange at 37 degrees C as a single kinetic pool in the presence of albumin (2%, w/v). However, we observed biphasic exchange kinetics with intact cells, indicating that cholesterol translocation from the inner to outer monolayers was rate-limiting in the exchange process. Approximately 50% of the cholesterol molecules were localized in each kinetic pool, independent of the cholesterol/phospholipid molar ratio in the cells and vesicles. A striking change in the kinetic parameters for cholesterol exchange occurred between 20 and 26 mol % cholesterol; for example, when the cholesterol/phospholipid molar ratio was decreased from 0.36 to 0.25, the half-time for equilibration of the two cholesterol pools at 37 degrees C decreased from 4.6 +/- 0.5 to 2.5 +/- 0.1 h. Phospholipid exchange rates were also enhanced on decreasing the membrane cholesterol content. The ability of cholesterol to modulate its own exchange rate, as well as that of phospholipids, is suggested to arise from the sterol's ability to regulate membrane lipid order. Extensive chemical modification of the membrane surface by cross-linking of some of the protein constituents with 1,4-phenylenedimaleimide decreased the cholesterol exchange rate. Depletion of membrane proteins by treatment of growing cultures with chloramphenicol increased the cholesterol exchange rate, possibly because of removal of some of the protein mass that may impede lipid translocation. The observations that phospholipid exchange was one order of magnitude slower than cholesterol exchange and that dimethyl sulfoxide, potassium thiocyanate, and potassium salicylate enhanced the cholesterol exchange rate are consistent with a mechanism involving lipid exchange by diffusion through the aqueous phase.     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.     

Proteomic and biochemical analyses of human B cell-derived exosomes. Potential implications for their function and multivesicular body formation

Exosomes are 60-100-nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane. Here we determined the protein and lipid compositions of highly purified human B cell-derived exosomes. Mass spectrometric analysis indicated the abundant presence of major histocompatibility complex (MHC) class I and class II, heat shock cognate 70, heat shock protein 90, integrin alpha 4, CD45, moesin, tubulin (alpha and beta), actin, G(i)alpha(2), and a multitude of other proteins. An alpha 4-integrin may direct B cell-derived exosomes to follicular dendritic cells, which were described previously as potential target cells. Clathrin, heat shock cognate 70, and heat shock protein 90 may be involved in protein sorting at multivesicular bodies. Exosomes were also enriched in cholesterol, sphingomyelin, and ganglioside GM3, lipids that are typically enriched in detergent-resistant membranes. Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-containing buffers. Multivesicular body-linked MHC class II was also resistant to CHAPS whereas plasma membrane-associated MHC class II was solubilized readily. Together, these data suggest that recruitment of membrane proteins from the limiting membranes into the internal vesicles of multivesicular bodies may involve their incorporation into tetraspanin-containing detergent-resistant membrane domains.     

Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium

Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed.     

Association of major histocompatibility complex II with cholesterol- and sphingolipid-rich membranes precedes peptide loading

Major histocompatibility complex class II protein (MHC II) molecules present antigenic peptides to CD4-positive T-cells. Efficient T cell stimulation requires association of MHC II with membrane microdomains organized by cholesterol and glycosphingolipids or by tetraspanins. Using detergent extraction at 37 degrees C combined with a modified flotation assay, we investigated the sequence of events leading to the association of MHC II with cholesterol- and glycosphingolipid-rich membranes (DRMs) that are distinct from tetraspanins. We find two stages of association of MHC II with DRMs. In stage one, complexes of MHC II and invariant chain, a chaperone involved in MHC II transport, enter DRMs in the Golgi stack. In early endosomes, these complexes are almost quantitatively associated with DRMs. Upon transport to late endocytic compartments, MHC II-bound invariant chain is stepwise proteolyzed to the MHC class II-associated invariant chain peptide (CLIP) that remains MHC II-bound and retains a preference for DRMs. At the transition between the two stages, CLIP is exchanged against processed antigens, and the resulting MHC II-peptide complexes are transported to the cell surface. In the second stage, MHC II shows a lower overall association with DRMs. However, surface MHC II molecules occupied with peptides that induce resistance to denaturation by SDS are enriched in DRMs relative to SDS-sensitive MHC II-peptide complexes. Likewise, MHC II molecules loaded with long-lived processing products of hen-egg lysozyme containing the immunodominant epitope 48-61 show a very high preference for DRMs. Thus after an initial mainly intracellular stage of high DRM association, MHC II moves to a second stage in which its preference for DRMs is modulated by bound peptides.     

Lateral diffusion of lipid probes in the surface membrane of human platelets. An electron-electron double resonance (ELDOR) study.

Electron-electron double resonance (ELDOR) techniques employing [14N], [15N] 16-Doxylstearate spin-label pairs have been used to measure the lateral diffusion constant, D, of lipids in the surface membrane of intact human blood platelets. For freshly prepared platelets, D is 1.0 X 10(-8) cm2/s at 37 degrees C and for platelets stored for 3 d at room temperature under accepted routine blood bank conditions, D is 2.6 X 10(-8) cm2/s at 37 degrees C. This is the first time that D in the surface membrane of platelets is reported. The marked increase in D for stored platelets may be attributed at least partly to loss of cholesterol during storage, suggesting a correlation between lipid lateral diffusion and cholesterol levels in cell membranes.     

A temperature-sensitive mutant of Newcastle disease virus defective in intracellular processing of fusion protein.

A temperature-sensitive mutant (ts3) of Newcastle disease virus was physiologically characterized. All major viral structural proteins were synthesized at the permissive (37 degrees C) and nonpermissive (42 degrees C) temperatures, but the fusion (F) glycoprotein was not cleaved at 42 degrees C. In immunocytochemical electron microscopy, the F protein was abundant in the rough endoplasmic reticulum but not in cytoplasmic membrane at 42 degrees C. Noninfectious hemagglutinating virus particles containing all major structural proteins except the F protein were released at 42 degrees C from infected cells. We concluded that the defect in ts3 resides in the intracellular processing of the F protein.