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PubMed1990

Characterization of the coated vesicle uncoating ATPase: tissue distribution, association with and activity on intact coated vesicles.

We have analyzed the uncoating process of clathrin-coated vesicles (CV) performed by an ATPase (UA; apparent molecular mass 70 kDa) prepared from various mammalian tissues. Our data show that this enzyme removes the clathrin coat from isolated, intact coated vesicles, as seen by sedimentation analysis on gels and also by electron microscopy. The isolated UA does not discriminate between CV from homologous or heterologous tissues. This finding implies that the brain-specific insertion in clathrin light chains cannot be essential for the binding of brain UA to target vesicles. Polyclonal antibodies were raised against UA and were found to inhibit UA activity. Immunoblotting of purified CV and immunoblotting of CV in situ indicate that a subpopulation of CV contains bound UA. However, most of the uncoating enzyme is not associated with coated structures in mammalian tissue culture cells. Our data support the hypothesis that the 70 kDa uncoating ATPase is responsible for the in vivo uncoating of coated vesicles.

Adenosine Triphosphatases↗

PubMed1991

Biocompatibility of extracorporeal circulation. In vitro comparison of heparin-coated and uncoated oxygenator circuits.

Oxygenator/tubing sets coated with endpoint-attached heparin were compared to uncoated sets in a dynamic model of extracorporeal circulation. Biocompatibility was assessed by evaluations of complement activation and platelet loss. The median concentration of C3 activation products increased gradually from 12 AU/ml at baseline to 65 AU/ml after 120 minutes in the uncoated sets and from 12 AU/ml to 19 AU/ml after 120 minutes in the coated sets (p less than 0.002). The median concentration of the terminal complement complex in the uncoated sets increased gradually from 3.1 AU/ml at baseline to 18.2 AU/ml after 120 minutes. In the coated sets the terminal complement complex reached a peak of 12.0 AU/ml at 15 minutes and returned to baseline values at 60 minutes. Median platelet loss at 120 minutes was 166 x 10(9) in the uncoated and 21 x 10(9) in the coated sets (p less than 0.01). Heparin coating thus improved biocompatibility by reducing both complement activation and platelet loss.

Biocompatible Materials↗

PubMed1985

ATP catalyzes the sequestration of clathrin during enzymatic uncoating.

ATP facilitates the sequestration of displaced triskelions by uncoating protein. In so doing, ATP is not hydrolyzed; nor does the concentration of ATP affect the equilibrium of this binding. However, the rates of both the binding of uncoating protein to clathrin and of their dissociation are greatly accelerated by ATP. These properties suggest that ATP acts catalytically to speed the capture of displaced triskelions by uncoating protein, as well as stoichiometrically in its hydrolysis to drive the displacement of triskelions from cages. The nucleotide specificity of this "catalytic" site for ATP on the uncoating protein is much less strict than that of the distinct "hydrolytic" site that drives the ATP-dependent displacement of triskelions from cages.

Adenosine Triphosphate↗

PubMed1983

Optical determination of coated and uncoated mineral fibers in lungs of subjects without professional exposure.

Coated and uncoated mineral fibers, more than 10 micron long, were counted by light microscopy in digested lung samples from 82 adult subjects without professional exposure. Up to about 150 coated and 12,500 uncoated fibers/g dry weight were found, without any correlation to sex, occupation and smoking habit. An association between counts of more than 1,000 fibers and ages over 50 was observed. The ratio of uncoated to coated fibers was very inconstant. Only the counts of both coated and uncoated fibers seem advisable for an optical evaluation of the possibly pathological mineral fiber burden in the lungs of unexposed persons.

Adolescent↗

PubMed1993

Complement activation during extracorporeal circulation. In vitro comparison of Duraflo II heparin-coated and uncoated oxygenator circuits.

The degree of complement activation during cardiopulmonary bypass is considered a valuable parameter of biocompatibility of the extracorporeal circuit. In an in vitro setting with a heart-lung machine primed with fresh whole blood and saline solution, the C3 activation products C3b, iC3b, and C3c and the terminal complement complex were measured in double-antibody enzyme immunosorbent assays. No differences were found between seven sets treated with Duraflo II heparin coating and seven uncoated sets after 2 hours of circulation. C3 activation products (expressed as median and 95% confidence intervals) increased from 4.5 AU (2.8 to 12.3 AU) to 16.5 AU (10.0 to 19.4 AU) in the uncoated sets (p = 0.02) and from 4.6 AU (2.2 to 5.8 AU) to 19.3 AU (3.5 to 27.1 AU) in the coated sets (p = 0.02). Terminal complement complex increased from 5.7 AU (2.7 to 11.3 AU) to 13.6 AU (8.2 to 17.8 AU) in the uncoated sets (p = 0.02) and from 7.9 AU (4.6 to 11.4 AU) to 17.3 AU (9.4 to 35.1 AU) in the coated sets (p = 0.02). A significant drop in thrombocyte levels was observed in both coated and uncoated sets. In a supplementary series, the sterilization process did not influence the results. Although Duraflo II heparin coating is considered highly effective in preventing coagulation, it did not prevent complement activation in the present in vitro study. We hypothesize that the mode by which the heparin molecule is bound to the surface may be essential to obtain effects on both coagulation and complement system.

Biocompatible Materials↗

PubMed1993

Nucleotide binding properties of bovine brain uncoating ATPase.

Many functions of the 70-kDa heat-shock proteins (hsp70s) appear to be regulated by bound nucleotide. In this study we examined the nucleotide binding properties of purified bovine brain uncoating ATPase, one of the constitutively expressed members of the hsp70 family. We found that uncoating ATPase purified by ATP-agarose column chromatography retained one ADP molecule bound per enzyme molecule which could not be removed by extensive dialysis. Since this bound ADP exchanged rapidly with free ADP or ATP, the inability to remove the bound nucleotide was not due to slow dissociation but rather to strong binding of the nucleotide to the uncoating ATPase. In confirmation of this view, equilibrium dialysis experiments suggested that the dissociation constants for both ADP and ATP were less than 0.1 microM. Schmid et al. (Schmid, S. L., Braell, W. A., and Rothman, J. E. (1985) J. Biol. Chem 260, 10057-10062) suggested that the uncoating ATPase had two sites for bound nucleotide, one specific for ATP and one binding both ATP and ATP analogues but not ADP. In contrast, we found that enzyme with bound ADP did not bind further adenosine 5'-(beta,gamma-imino)triphosphate or dATP, nor did more than one ATP molecule bind per enzyme even in 200 microM free ATP. These results strongly suggest that the enzyme has only one binding site for nucleotide. During steady-state ATP hydrolysis, 85% of the bound nucleotide at this site was determined to be ATP and 15% ADP; this is consistent with the rate of ADP release determined in the exchange experiments noted above, where ADP release was found to be six times faster than the overall rate of ATP hydrolysis.

Adenosine Diphosphate↗

PubMed2004

Sirolimus-eluting vs uncoated stents for prevention of restenosis in small coronary arteries: a randomized trial.

CONTEXT: Percutaneous coronary revascularization of small vessels is associated with a high restenosis rate. Sirolimus-eluting stents reduce restenosis in simple and previously untreated lesions of large coronary arteries, but their outcomes in small vessels have not been adequately investigated. OBJECTIVE: To determine whether sirolimus-eluting stents are associated with a reduced 8-month rate of angiographic restenosis in comparison with an uncoated stent. DESIGN, SETTING, AND PATIENTS: This was a randomized, multicenter, single-blind, prospective trial performed with 257 patients undergoing percutaneous coronary revascularization for ischemic heart disease, and who had a previously untreated atherosclerotic lesion located in a small segment with a diameter of 2.75 mm or less, in 20 Italian centers between August 2002 and December 2003. INTERVENTION: Patients were randomly assigned to receive a sirolimus-eluting stent (129 patients) or an uncoated stent having an identical architecture and radiographic appearance (128 patients). MAIN OUTCOME MEASURES: The primary end point was the 8-month binary in-segment restenosis rate; secondary end points included procedural success and the 8-month rate of major adverse cardiac and cerebrovascular events. RESULTS: The mean (SD) reference diameter of the treated segment was 2.2 (0.28) mm; the lesion length, 11.84 (6.15) mm. After 8 months, the binary in-segment restenosis rate was 53.1% (60/113) in the patients receiving an uncoated stent and 9.8% (12/123) in those receiving a sirolimus-eluting stent (relative risk [RR], 0.18; 95% confidence interval [CI], 0.10-0.32; P<.001). Fewer patients randomized to sirolimus-eluting stents experienced major adverse cardiac events (12/129 [9.3%] vs 40/128 [31.3%]; RR, 0.30; 95% CI, 0.15-0.55; P<.001) mainly because of a reduction in target lesion revascularization (9/129 [7%] vs 27/128 [21.1%]; RR, 0.33; 95% CI, 0.14-0.70; P = .002) and myocardial infarction (2/129 [1.6%] vs 10/129 [7.8%]; RR, 0.20; 95% CI, 0.01-0.93; P = .04). CONCLUSION: The use of sirolimus-eluting stents to treat atherosclerotic lesions in small coronary arteries reduces restenosis and may also reduce major adverse cardiac events.

Aged↗

PubMed2004

Separation of fluorescein isothiocyanate-labeled amines by microchip electrophoresis in uncoated and polyvinyl alcohol-coated glass chips using water and dimethyl sulfoxide as solvents of background electrolyte.

On-chip capillary electrophoresis with uncoated and polyvinyl alcohol-coated glass channels in aqueous and nonaqueous dimethyl sulfoxide (DMSO) background electrolyte (BGE) solutions was applied in the separation of five amines derivatized with fluorescein-5-isothiocyanate. In aqueous BGE at pH 9.2, baseline separation of the analytes was not achieved on uncoated glass chips, but the separation was clearly improved when the chip channels were coated with polyvinyl alcohol (PVA). Separation was successful in nonaqueous DMSO electrolyte solution containing ammonium acetate and sodium methoxide, on both uncoated and PVA-coated glass microchips. The differences in the pK(a) values of analytes were probably amplified in DMSO, and all five analytes were at least partly dissociated and were separated. Because the viscosity of DMSO is higher than that of water, the migration times were longer in DMSO.

Amines↗

PubMed1996

Analyses of the role of structural changes in the regulation of uncoating and assembly of alphavirus cores.

In the late stages of alphavirus multiplication virus cores accumulate in the cytoplasm as stable structures, whereas they are unstable during the early stages of infection. Three types of explanation can be put forward to understand this phenomenon: (1) A cellular uncoating process is active early which is inactivated later, (2) the core structure differs between stable cores which accumulate and labile cores which are dissociated, (3) both mechanisms cooperate. A model based exclusively on the first principle involving core disassembly by cellular ribosomes has been proposed, but structural changes of the core may cooperate with the cellular uncoating process. We have therefore isolated cores of the Sindbis alphavirus from the cellular cytoplasm, from virus particles (vi-cores), and from low-pH-treated virus particles. Comparative analyses of the structure of these cores, using crosslinking and proteolytic digestion, and of the stability of the cores in the presence of ribosomes in vitro were performed. The structural comparisons indicate that the interactions between the molecular components of these cores are very similar and probably identical. In the presence of ribosomes vi-cores were slightly more stable than the two other types of cores. Evidence for a cellular uncoating mechanism is furnished by experiments which analyze the stability of cores in the presence of postmitochondrial cytoplasm or of 60 S ribosomal subunits derived from either uninfected or Sindbis virus-infected cells. The results obtained indicate that structural alterations of the core do not play a role in the regulation of disassembly and assembly of alphavirus cores.

Animals↗

PubMed1997

Multiple mechanisms for the inhibition of entry and uncoating of superinfecting Semliki Forest virus.

Recombinant Semliki Forest viruses (SFV) that express one or none of the viral structural proteins were used to infect cells and to analyze the fate of incoming superinfecting wild-type viruses. It was found that in addition to the previously described block in replication that superinfecting viruses encounter within 15 min of infection, other mechanisms of superinfection inhibition occurred at later times. Over a 6-hr infection period, inhibition was seen in binding of virus to the cell surface, in acid-activated penetration into the cytoplasm, and in uncoating of nucleocapsids. For each of these processes, the inhibitory mechanism was investigated. In summary, we found that infection evoked several independent mechanisms for blocking the entry and uncoating of superinfecting viruses. The results also offered new insights into the normal processes of penetration and uncoating of SFV.

Animals↗

PubMed1990

Role of pH in syncytium induction and genome uncoating of avian infectious bronchitis coronavirus (IBV).

Syncytium formation induced in Vero cells by IBV-Beaudette was optimal at pH 6.7 and absent at pH 7.5. Reduction of IBV-Beaudette replication by ammonium chloride, which raises the pH in endosomes, further indicated that uncoating of the virus genome probably occurred within the acidic environment of endosomes. However, this would not appear to apply to all IBV strains, since one was unaffected by the drug. It would appear that some strains of IBV have the capacity to fuse with the plasma membrane at neutral pH, thereby leading to genome uncoating at that site. Other strains of IBV might also uncoat at the plasma membrane when the extracellular environment is slightly less than pH7.

Ammonium Chloride↗

PubMed1994

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy.

HeLa metaphase chromosomes were examined by means of "in lens" field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Chromosomes, Human↗

PubMed1998

Inhibition of foot and mouth disease virus (FMDV) uncoating by a plant-derived peptide isolated from Melia azedarach L leaves.

Meliacine (MA), a peptide isolated from leaves of the high plant Melia azedarach L inhibited the multiplication of foot and mouth disease virus (FMDV) in BHK-21 cells. In this report, we establish that the MA-inhibitable process takes place within the first hour of the viral reproductive cycle. MA had no virucidal effect and did not affect adsorption and penetration of the virus in cells. In experiments with neutral red-labeled virus, it was found that MA significantly suppressed the development of photoresistance of the virus in infected cells. In untreated cultures nearly all virus which adsorbed to cells was uncoated within 1 h at 37 degrees C, whereas in treated cultures, even after 3 h only 3% of the virus was uncoated. Labeling of BHK-21 cells with acridine orange showed that MA affects the pH of intracellular acidic vesicles. Therefore, it is concluded that MA prevents the process of uncoating of FMDV in BHK-21 cells by inhibiting vacuolar acidification.

Animals↗

PubMed2005

Microstructural analysis of implant-bone interface of hydroxyapatite-coated and uncoated Schanz screws.

The aim of the study was to compare the pin-bone interface microstructural characteristics of hydroxyapatite-coated (HAC) and stainless steel Schanz screws after 2, 4 and 6 months of implantation in a sheep model. The microstructure and composition of the hydroxyapatite coating were analyzed using scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. Twelve coated and 12 uncoated screws were implanted into both femora of three sheep, each sheep receiving eight screws. Specimens of polished bone with screws were examined with SEM and light microscope for morphometric analyses. The HAC was approx. 40 microm thick, the grain size ranged from 5 to 40 microm, with pores less than 20 microm. The atomic ratio of Ca/P was 1.62. SEM showed that the bone-implant contact was better with HAC than with uncoated implants. The ingrowth of the bone in the HAC was clearly seen. Morphometric analysis showed good bone-implant contact in 65.1 (+/-24.6)% in the HAC and 32.0 (+/-23.3)% in the uncoated group (p<0.001). Although the percentage of good contact increased with time for both groups, it was significantly higher for HAC screws. Our investigation demonstrated a time dependent improvement of implant-bone contact of the HAC compared to standard stainless steel implants in the chosen experimental conditions.

Animals↗

PubMed1990

Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LCa to stimulate ATP hydrolysis.

Uncoating of clathrin-coated vesicles is mediated by the heat shock cognate protein, hsc70, and requires clathrin light chains (LCa and LCb) and ATP hydrolysis. We demonstrate that purified light chains and synthetic peptides derived from their sequences bind hsc70 to stimulate ATP hydrolysis. LCa is more effective than LCb in stimulating hsc70 ATPase and in inhibiting clathrin uncoating by hsc70. These differences correlate with high sequence divergence in the proline- and glycine-rich region (residues 47-71) that forms the hsc70 binding site. For LCa, but not LCb, this region undergoes reversible conformational changes upon perturbation of the ionic strength or the calcium ion concentration. Our results show that LCa is more important for interactions with hsc70 than is LCb and suggest a model in which the LCa conformation regulates coated vesicle uncoating.

Adenosine Triphosphatases↗

PubMed1994

Histomorphometry of hydroxyapatite coated and uncoated porous titanium bone implants.

Two different groups of hydroxyapatite (HA) coated and uncoated porous titanium implants, 250-350 microns and 500-700 microns diameter beads, were press-fitted into femoral canine cancellous bone. After 12 wks, the dogs were killed and histomorphometric backscattered electron microscopy studies were carried out. Comparing HA coated versus uncoated implants in the 250-350 microns bead diameter group, the percentage of bone (P = 0.01) and bone index (P = 0.01) were higher in the HA coated implants. Comparing HA coated versus uncoated implants in the 500-700 microns bead diameter group, bone ingrowth (P = 0.01) and bone depth penetration (P = 0.008) were higher in HA coated samples. It can be concluded that the HA coating was an effective method for improving bone formation and ingrowth in the porous implants.

Alloys↗

PubMed2006

Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging.

Differences among models in the temporal evolution of ischemia after middle cerebral artery occlusion (MCAO) in rats may considerably influence the results of experimental treatment studies. Using diffusion and perfusion imaging, we compared the spatiotemporal evolution of ischemia in Sprague-Dawley rats after permanent MCAO (pMCAO) with different types of sutures. Male Sprague-Dawley rats were randomly assigned to pMCAO produced with either 4-0 silicone coated (n=8), or 3-0 uncoated monofilaments (n=8). Serial determination of quantitative cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) maps were performed up to 3 h after pMCAO. Lesion volumes were calculated by using previously validated thresholds and correlated with infarct volume corrected for edema defined by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 24 h after MCAO. The ADC/CBF-defined mismatch volume in the 4-0 coated suture model was present significantly longer (up to 120 min) compared to the uncoated 3-0 suture model (30 min). The TTC-derived infarct volume was significantly larger in the coated model (290.3+/-32.8 mm(3)) relative to the uncoated model (252.3+/-34.6 mm(3)). This study demonstrates that the type of suture may significantly influence the spatiotemporal evolution of the ADC/CBF-mismatch as well as the final infarct volume. These inter-model variations must be taken into account when assessing new therapeutic approaches on ischemic lesion evolution in the rat MCAO model.

Animals↗

PubMed2004

HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion.

We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells. This assay involves the use of virions containing beta-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of beta-lactamase. Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr. Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating. We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells. Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry. Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane.

Cell Line↗