Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a past-immunity serum sample gave an average diffusion coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7 x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed with acute phase or past-immunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and past-immunity sera. FCS measurement of VLP-immune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.     

Properties of baculovirus particles displaying GFP analyzed by fluorescence correlation spectroscopy

Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89 +/- 0.74) x 10(-8) cm2s(-1) and an apparent hydrodynamic radius of 83.35 +/- 21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.57 x 10(-7) cm2s(-1)), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.     

Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium

In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular crowding reduces to a similar extent the diffusion of small solutes and macromolecules: measurement by fluorescence correlation spectroscopy

Aqueous environments in living cells are crowded, with up to >50 wt% small and macromolecule-size solutes. We investigated quantitatively one important consequence of molecular crowding--reduced diffusion of biologically important solutes. Fluorescence correlation spectroscopy (FCS) was used to measure the diffusion of a series of fluorescent small solutes and macromolecules. In water, diffusion coefficients (D(o)w) were (in cm2/s x 10(-8)): rhodamine green (270), albumin (52), dextrans (75, 10 kDa; 10, 500 kDa), double-stranded DNAs (96, 20 bp; 10, 1 kb; 3.4, 4.5 kb) and polystyrene nanospheres (5.4, 20 nm diameter; 2.3, 100 nm). Aqueous-phase diffusion (Dw) in solutions crowded with Ficoll-70 (0-60 wt%) was reduced by up to 650-fold in an exponential manner: Dw = D(o)w exp (-[C]/[C]exp), where [C]exp is the concentration (in wt%) of crowding agent reducing D(o)w by 63%. FCS data for all solutes and Ficoll-70 concentrations fitted well to a model of single-component, simple (non-anomalous) diffusion. Interestingly [C]exp were nearly identical (11+/-2 wt%, SD) for diffusion of the very different types of macromolecules in Ficoll-70 solutions. However, [C]exp was dependent on the nature of the crowding agent: for example, [C]exp for diffusion of rhodamine green was 30 wt% for glycerol and 16 wt% for 500 kDa dextran. Our results indicate that molecular crowding can greatly reduce aqueous-phase diffusion of biologically important macromolecules, and demonstrate a previously unrecognized insensitivity of crowding effects on the size and characteristics of the diffusing species.     

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.     

Cryopreservation and in vitro expansion of chondroprogenitor cells isolated from the superficial zone of articular cartilage

Understanding the proliferation mechanisms of chondroprogenitor cells and their influence on cell differentiation is crucial in order to develop large-scale expansion processes for tissue engineering applications. Proliferation control mechanisms were mainly attributed to substrate limitation and cell-cell contact inhibition. The limiting substrates were found to be components of the FCS, with an optimal proliferation rate achieved in the presence of 40% FCS. In addition, the medium supply rate was found to be essential in reducing substrate limitation. In terms of FCS, 10 microL FCS cm(-2) h(-1) was the threshold feed rate required to prevent substrate limitation. Above this rate, maximum cell densities of 5.3 x 10(5) cells/cm2 were achieved, representing a 53-fold expansion. To reduce the need for high supply rates, the effect of specific growth factors was also investigated. Cell densities of 3.3 x 10(5) cells/cm2 were achieved in batch cultures using 40% FCS and 1 ng/mL TGF-beta1. Chondroprogenitor cells were expanded in this medium up to three passages without compromising their ability to differentiate and produce cartilage-like matrix in pellet cultures. In addition to substrate limitation, cell-cell contact, even at very sparse subconfluent densities, appeared capable of exerting some degree of growth inhibition. The cells exhibited deceleratory growth kinetics, characterized by a decrease of specific growth rates over time.     

UV-Fluorescence correlation spectroscopy of 2-aminopurine

We have built a fluorescence correlation spectroscopy (FCS) microscope for ultraviolet excitation (280-300 nm) and emission. With UV excitation the fluorescence of 'natural fluorophores' such as the modified nucleotide 2-aminopurine can be analyzed. The sensitivity of a natural fluorophore toward conformational changes can reveal dynamics in biomolecules. UV-FCS is well suited for detection of intensity fluctuations related to such conformational dynamics. Here we show UV-FCS measured on p-Quarterphenyl and on 2-aminopurine (2-AP). The triplet state rate constants and the excitation cross section for 2-AP were estimated to k23 = 1 x 10(6) s(-1), k31 = 3 x 10(5) s(-1), and sigma(exc) = 2 x 10(-17) cm2.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

A new procedure for the cryopreservation of equine embryos

Early equine blastocysts and blastocysts were collected nonsurgically at six days post-ovulation. Thirty-two embryos were randomly assigned to a 2x2 factorial design. Factors were: 1) 0.5-ml straws or 1-ml glass ampules; and 2) plunging into liquid nitrogen (IN(2)) at -33 C or -38 C. Cryoprotectant, 10% glycerol in PBS plus 5% fetal calf serum (FCS) was added in two steps, 5% then 10%. Embryos were cooled at 4 C/min to -6 C and then seeded, 0.3 C/min to -30 or -35 C and 0.1 C/min to -33 or -38 C. Samples were thawed in 37 C water and glycerol removed in six steps, 10 min per step. Embryo quality and stage of development were evaluated prior to freezing, immediately post-thaw and after 24 h culture in Ham's F10 with 5% FCS. The mean post-thaw quality of embryos plunged at -33 C was superior (P<0.05) to that of embryos plunged at -38 C (2.0 vs 2.9). Embryos frozen in ampules and plunged at -38 C were of poorer quality (P<0.05) than those frozen in ampules and plunged at -33 C or frozen in straws and plunged at -33 C. After 24 h of culture, more embryos developed if frozen in straws compared to ampules, and plunging at -33 C resulted in higher quality embryos than plunging at -38 C. In Experiment 2, 23 embryos were packaged in straws and plunged at -33 C as described in Experiment 1. Six of the 23 surgically transferred frozen embryos were degenerate at thawing and the remaining 17 surgically transferred were via flank incision. Pregnancy rate at 50 days post-ovulation was 53% (nine of 17). Early blastocysts resulted in a higher (P<0.05) pregnancy rate (8 10 , 80%) than expanded blastocysts (1 7 , 14%).     

Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.     

Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen processing and intracellular Ia. Possible roles of endocytosis and protein synthesis in Ia function

Anti-I-A mAb and monovalent Fab fragments were used to explore the cellular distribution and endocytosis of I-A in peritoneal exudate cells (PEC) and TA3 B lymphoma-hybridoma cells. TA3 cells contained 1.6 x 10(5) I-A sites/cell, 22 to 35% of which were intracellular. This intracellular pool was cycloheximide resistant. PEC contained 1.8 x 10(5) I-A sites/cell, 25 to 40% of which were intracellular. Upon adherence, however, the intracellular pool of I-A in PEC dropped to 2 to 11% of the total cellular I-A. Ag processing by TA3 cells was unaffected 3 h after abrogation of protein synthesis with cycloheximide, suggesting that newly synthesized I-A is not necessary for Ag processing in TA3 cells (post-synthetic processing and transport of I-A to the plasma membrane were complete by 2 h in TA3 cells with or without cycloheximide, as assessed by sequential immunoprecipitation of surface and intracellular I-A). In adherent PEC, however, cycloheximide markedly inhibited Ag processing, suggesting depletion of factors necessary for Ag processing. Ag processing may involve binding of processed Ag peptides to intracellular Ia derived to varying degrees from both endocytosis and new biosynthesis. To explore the possibility of I-A recycling, I-A endocytosis was demonstrated using mAb and monovalent Fab probes; internalization occurred within 5 min and peaked by 10 to 15 min with 15 to 35% of bound antibody in an intracellular compartment, resistant to an acid wash. Subcellular density gradient fractionation demonstrated that I-A and transferrin were processed exclusively in an endosomal fraction of relatively light density, whereas ligands of the mannose receptor were processed in light endosomes and in a distinct, denser population of endosomes, and accumulated in lysosomes. Thus, I-A appears to be internalized into a specific population of endosomes that may play a central role in Ag processing.     

Diffusion in the endoplasmic reticulum of an aquaporin-2 mutant causing human nephrogenic diabetes insipidus

Mutations in the aquaporin-2 (AQP2) water channel cause the hereditary renal disease nephrogenic diabetes insipidus (NDI). The missense mutation AQP2-T126M causes human recessive NDI by retention at the endoplasmic reticulum (ER) of renal epithelial cells. To determine whether the ER retention of AQP2-T126M is due to relative immobilization in the ER, we measured by fluorescence recovery after photobleaching the intramembrane mobility of green fluorescent protein (GFP) chimeras containing human wild-type and mutant AQP2. In transfected LLC-PK1 renal epithelial cells, GFP-labeled AQP2-T126M was localized to the ER, and wild-type AQP2 to endosomes and the plasma membrane; both were localized to the ER after brefeldin A treatment. Photobleaching with image detection indicated that the GFP-AQP2 chimeras were freely mobile throughout the ER. Quantitative spot photobleaching revealed a diffusion-dependent irreversible process whose recovery depended on spot size and was abolished by paraformaldehyde fixation. In addition, a novel slow reversible fluorescence recovery (t(12) approximately 2 s) was characterized whose recovery was independent of spot size and not affected by fixation. AQP2 translational diffusion in the ER was not slowed by the T126M mutation; diffusion coefficients were (in cm(2)/s x 10(-)10) 2.6 +/- 0.5 (wild-type) and 3.0 +/- 0.4 (T126M). Much faster diffusion was found for a lipid probe (diOC(4)(3), 2.7 x 10(-)8 cm(2)/s) in the ER membrane and for unconjugated GFP in the aqueous ER lumen (6 x 10(-)8 cm(2)/s). ER diffusion of GFP-T126M was not significantly affected by up-regulation of molecular chaperones, cAMP activation, or actin filament disruption. ATP depletion by 2-deoxyglucose and azide resulted in comparable slowing/immobilization of wild-type and T126M AQP2. These results indicate that the ER retention of AQP2-T126M does not result from restricted or slowed mobility and suggest that the majority of AQP2-T126M is not aggregated or bound to slowly moving membrane proteins.     

Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.     

The late endosomal p14–MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14–MP1 (LAMTOR2/3) complex in FA dynamics. p14–MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end–directed traffic of p14–MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14–MP1 scaffold complex to the vicinity of FAs.     

Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae.

35S-labeled killer toxin protein bound to cells of sensitive Saccharomyces cerevisiae S14a. Strains that were resistant to toxin through mutation in the nuclear genes kre1 kre2 bound toxin only weakly. Non-radioactive toxin competed effectively with 35S-labeled toxin for binding to S14a, but did not compete significantly in the binding to mutant kre1-1. This implied that binding to kre1-1 was nonspecific. A Scatchard analysis of the specific binding to S14a gave a linear plot, with an association constant of 2.9 x 10(6) M-1 and a receptor number of 1.1 x 10(7) per cell. Killer toxin receptors were solubilized from the cell wall by zymolyase digestion. Soluble, non-dialyzable cell wall digest from S14a competed with sensitive yeast cells for 35S-labeled toxin binding and reduced toxin-dependent killing of a sensitive strain. Wall digest from kre1-1 competed only weakly for toxin binding with sensitive cells and caused little reduction of toxin-dependent killing. Although the abundant (1.1 x 10(7) per cell) receptor appeared necessary for toxin action, as few as 2.8 x 10(4) toxin molecules were necessary to kill a sensitive cell of S14a. The kinetics killing of S14a suggested that some component was saturated with toxin at a concentration 50-fold lower than that needed to saturate the wall receptor.     

Receptor-mediated uptake and processing of vitamin D-binding protein in human B-lymphoid cells.

Vitamin D-binding protein (DBP), a member of a multigene family including alpha-fetoprotein (AFP) and albumin, is a serum glycoprotein that reversibly binds and transports vitamin D and its metabolites to target cells. In this work, we demonstrate that normal and malignant human B-lymphocytes specifically bind and internalize DBP. Radioiodinated DBP (125I-DBP) was used to follow the uptake of the protein by Raji cells, a human pre-B-lymphoma cell line. Time course studies of DBP uptake by these cells exhibited a saturable profile at both 4 and 37 degrees C. The binding saturation curve obtained by incubating Raji cells at 4 degrees C with different concentrations (1.5 nM to 1.5 microM) of 125I-DBP showed two saturation plateaus; Scatchard analysis showed the presence of two groups of receptor sites with a Kd1 of 2.04 x 10(-7) M (n1 = 42,161 +/- 4,336 sites/cell) and a Kd2 of 1.01 x 10(-6) M (n2 = 198,000 +/- 48,000 sites/cell). After incubation of Raji cells at 37 degrees C with both fluorescein isothiocyanate (FITC) and horseradish peroxidase conjugates, DBP was internalized and could be localized in the cytoplasm. DBP-horseradish peroxidase conjugates were used to follow the uptake and to determine the endocytic pathway of the protein in Raji cells. The initial steps, contrary to those observed for AFP, did not apparently involve coated pits and vesicles. Small vesicles (approximately 50-60 nm) with electron-dense DBP-horseradish peroxidase reaction products were observed that could fuse with large endosomes. These endosomes appeared dispersed in the cytoplasm with some preferential localization in the Golgi centrosphere region. Pulse-chase experiments showed that only 10% of the uptaken protein was released in a nondegraded form. Accordingly, most DBP molecules accumulated in endosomes should be degraded in lysosomes, instead of being recycled back to the surface, as in the case of AFP. Contrary to malignant B-cells (Raji), the uptake ability for DBP of normal quiescent B-lymphocytes was very low. Specific binding and internalization of DBP-FITC by these cells were observed following mitogen-induced activation. Significant values of uptake were obtained at 37 degrees C after 72 h of incubation in the presence of pokeweed mitogen. The binding of DBP-FITC was partially inhibited in the presence of an excess of unlabeled protein. Taken together, the actual results suggest that DBP receptors are constitutively expressed by malignant B-cells and in a transitory form by normal B-lymphocytes undergoing mitogen-induced activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of proliferation and differentiation of myoblasts derived from adult mouse skeletal muscle by specific isoforms of PDGF

The expression of receptors and the mitogenic response to PDGF by C2 myoblasts, derived from adult mouse skeletal muscle, was investigated. Employing 125I-PDGF binding assays, we showed that the cells exhibit high level binding of PDGF-BB (approximately 165 x 10(3) molecules/cell at saturation) and much lower binding of the PDGF-AA and PDGF-AB (6-12 x 10(3) molecules/cell at saturation). This indicates that the C2 myoblasts express high levels of PDGF receptor beta-subunits and low levels of alpha-subunits. PDGF-BB enhances the proliferation of C2 cells maintained in 2% FCS by about fivefold. PDGF-AB had a moderate effect on cell proliferation (less than twofold) and PDGF-AA had no effect. Inverse effects of PDGF isoforms on the frequency of differentiated myoblasts were observed; the frequency of myosin-positive cells was reduced in the presence of PDGF-BB while PDGF-AA and PDGF-AB had no effect. PDGF may thus act to increase the number of myoblasts that participate in muscle regeneration following muscle trauma by stimulating the proliferation and by inhibiting the differentiation of myogenic cells.     

Modifications of LDL-receptor-mediated endocytosis rates in CEM lymphoblastic cells grown in lipoprotein-depleted fetal calf serum

The efficiency of supplying cholesterol by the LDL endocytic pathway of lymphoblastic T CEM cells was compared when incubated in the presence of either fetal calf serum (FCS) or lipoprotein-depleted fetal calf serum (LDFCS). In the presence of FCS, there were 8600 +/- 2000 LDL receptors/cell with a Kd of (2.2 +/- 0.8).10(-8) M and a receptor cycling time of about 7 min; about 90% of the internalized LDL was degraded. LDL degradation produced 98% of total cellular cholesterol and only 2% came from endogenous synthesis. The absence of LDL in the culture medium of lymphoblastic CEM cells deeply modified certain metabolic and structural characteristics of the cells. Their cholesterol content decreased; the total number of LDL receptors increased 6-fold, whereas their affinity for the ligand decreased by the same factor (Kd = (1.2 +/- 0.2).10(-7) M); the receptor cycling time increased 3-fold. Finally, LDL degraded by cholesterol-depleted CEM cells amounted to about 40% of that degraded by untreated CEM cells.     

Dynamics of beta2-adrenergic receptor-ligand complexes on living cells

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.