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PubMed2003

Intense inflammatory reaction to heparin polymer coated intravascular Palmaz stents in porcine arteries compared to uncoated Palmaz stents.

The objective of this study was to evaluate the efficacy of heparin-polymer-coated intravascular stents in the reduction of vessel stenosis. Three types of coatings for Palmaz stents were tested: 1) heparin covalently bound to a polyethylene oxide (Hp-PEO) tether; 2) heparin copolymerized with ethylene vinyl acetate (Hp-Elvax) and 3) Elvax alone. Polymer-coated stents and uncoated controls were deployed in the external iliac arteries following endothelial injury in 18 swine. The animals were maintained on an atherogenic diet and examined by angiography at 6 and 12 weeks. The stented segments were then harvested for histopathologic analysis. Both types of heparin-coated stents resulted in increased luminal narrowing as compared to the contralateral uncoated stents. At 6 weeks, average luminal stenosis was 48% for Hp-PEO stents vs 35% for uncoated stents (p <0.05). At 12 weeks, average luminal stenosis was 36% for Hp-PEO stents vs 26% for uncoated stents (p = NS). For Hp-Elvax stents, the average stenosis was 58% vs 33% for uncoated controls (p < 0.05) at 6 weeks and 47% vs 19% for uncoated controls (p <0.05) at 12 weeks. There was no significant difference between Hp-Elvax stents and Elvax stents (p = NS). Increased luminal narrowing in coated stents was primarily secondary to a marked inflammatory response. Heparin-polyethylene oxide and heparin-ethylene vinyl acetate-coated stents resulted in increased luminal narrowing as compared with uncoated stents, due to a marked inflammatory response.

Animals↗

PubMed1994

Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues.

The interactions of the 70-kDa heat-shock proteins (hsp70s) with their protein substrates appear to be regulated by bound nucleotide. Previous work has shown that the nucleotide binding site of the bovine brain uncoating ATPase, a constitutive member of the hsp70 family, crystallographically resembles the nucleotide binding site of actin and, like actin, the uncoating ATPase has a strongly bound ADP which cannot be removed by dialysis or treatment with ethylenediaminetetraacetic acid (EDTA). This suggests that, like the bound nucleotide of actin, it may be required for the enzyme to retain its native structure. In this study, the strongly bound ADP was removed by first replacing it with 5'-adenylyl imidodiphosphate (AMP-PNP) and then removing the bound AMP-PNP by dialysis. Following this treatment, more than 95% of the uncoating ATPase becomes nucleotide-free. The nucleotide-free uncoating ATPase retains its ability to bind and hydrolyze ATP and to uncoat clathrin-coated vesicles, even after 10 days of storage at 4 degrees C. Therefore, in contrast to actin, the bound nucleotide of the uncoating ATPase is not required to prevent denaturation of the enzyme. Using nucleotide-free uncoating ATPase, we were able to accurately measure the dissociation constants of ATP, ADP, and the nucleotide analogues AMP-PNP and 2'-deoxyadenosine 5'-triphosphate (dATP). The dissociation constants of both ATP and ADP are about 10(-8) M, more than 1-2 orders of magnitude stronger than previously reported, while AMP-PNP and dATP bind 2-3 orders of magnitude more weakly than ATP.

Adenosine Diphosphate↗

PubMed1975

Elution and uncoating of Coxsackievirus B3 by isolated HeLa cell plasma membranes.

Plasma membranes isolated from HeLa cells on discontinuous sucrose gradients were assayed for their capacity to elute and uncoat coxsackievirus B3 at 37 C. Because the viral receptors are limited to the surface of HeLa cells, the addition of radioactively labeled virus to the cells prior to cell homogenization provided a useful marker for locating the plasma membranes during the fractionation procedure. Four bands were formed on the discontinuous sucrose gradients with approximately 70% or more of the membrane-associated viral label being recovered in the most dense bands, designated as bands 3 and 4. Bands 3 and 4 also possessed the plasma membrane marker enzymes, Na+, K+ adenosine triphosphatase and 5'-nucleotidase and revealed typical structures characteristic of plasma membranes as revealed by electron microscopy. Pelleted and washed membranes from band 3 both eluted and uncoated B3 32P-labeled virus, whereas membranes from band 4 eluted virus but failed to uncoat it. The membranes from band 4 were shown to inhibit the viral uncoating activity when mixed with membranes of band 3. Characteristically, unfractionated homogenates of cell membranes eluted but did not uncoat virus. The finding of a naturally occurring inhibitor of virus uncoating provides for the first time a way to distinguish between the membrane activities of virus elution and virus uncoating. The inhibitor remains to be characterized.

Adenosine Triphosphatases↗

PubMedDate not supplied

Effect of beta-chemokines on human immunodeficiency virus type 1 replication, binding, uncoating, and CCR5 receptor expression in human monocyte-derived macrophages.

OBJECTIVES: We examined the effect and time of addition of beta-chemokines on human immunodeficiency virus type 1 (HIV-1) replication, binding, and uncoating in human macrophages and measured CCR5 receptor expression during virus binding and uncoating. METHODS: Macrophages were treated with beta-chemokines before infection, at infection, or postinfection, and virus replication was determined by p24 antigen level. Binding and uncoating of 35[S]-methionine-labeled HIV-1 was measured. CCR5 expression was determined by flow cytometry. RESULTS: The beta-chemokines potently inhibited virus replication. The strongest inhibition occurred when cultures were pretreated and maintained with beta-chemokines. Beta-chemokines also caused strong inhibition of viral uncoating and a considerable decrease in CCR5 expression during uncoating. CONCLUSIONS: CCR5 receptors appear to be internalized and recycled to the cell surfaces during HIV entry. The down-regulation of CCR5 expression by beta-chemokines during virus uncoating probably accounts for the reduction in virus uncoating (entry) and hence in virus replication.

CD4 Antigens↗

PubMed2003

Biological assessment of the bone-screw interface after insertion of uncoated and hydroxyapatite-coated pedicular screws in the osteopenic sheep.

The sheep seems to be a promising model of osteoporosis and biomaterial osteointegration in osteopenic bone. The long-term ovariectomized sheep model was used for the biological investigation of bone healing around uncoated and hydroxyapatite (HA)-coated pedicle screws in osteopenic bone. Four sheep were ovariectomized and four sheep were sham-operated. Twenty-four months after surgery, the animals were implanted with uncoated and HA-coated stainless steel screws in the lumbar vertebral pedicles. Four months later, bone-to-implant contact, bone ingrowth, and bone hardness were measured around screws. Uncoated stainless steel presented significantly (p < 0.0005) lower bone-to-implant contact in healthy and osteopenic bone compared with HA-coated stainless steel. HA significantly improved bone ingrowth in healthy bone (p < 0.05) compared with uncoated stainless steel. Osteopenia significantly (p < 0.05) reduced the area of bone ingrowth around the screw threads for both types of implants. In the inner thread area, bone microhardness significantly increased (p < 0.05) in HA-coated surface versus uncoated for healthy and osteopenic bone. HA coating significantly enhances bone-to-implant contact also in osteopenic bone in comparison with uncoated stainless steel surfaces. Bone ingrowth and mineralization are ameliorated by the osteoconductive HA coating. However, osteopenia seems to greatly influence bone ingrowth processes around the implanted screws regardless of the characteristics of the material surface.

Animals↗

PubMed2001

Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin.

We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxilin prevented binding to Hsc70 in vitro and injecting this mutant protein inhibited CCV uncoating in vivo, demonstrating that the interaction of auxilin with Hsc70 is critical for CCV uncoating. These studies establish that auxilin and Hsc70 participate in synaptic vesicle recycling in neurons and that an interaction between these proteins is required for CCV uncoating.

Adaptor Proteins, Vesicular Transport↗

PubMed1996

Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins.

The uncoating of clathrin-coated vesicles can be mediated in vitro by the 'uncoating ATPase' that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Although we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.

Adaptor Proteins, Vesicular Transport↗

PubMed1997

Biomechanical, scanning electron microscopy, and microhardness analyses of the bone-pin interface in hydroxyapatite coated versus uncoated pins.

OBJECTIVE: To evaluate the bone-pin interface in hydroxyapatite coated versus uncoated pins. DESIGN: Eighty-four bicylindrical stainless steel external fixation pins were implanted in a test group of 14 sheep. One-half of the pins were coated with hydroxyapatite and the rest remained uncoated. INTERVENTION: Six coated pins were implanted in the left tibia of seven sheep, and six uncoated pins were implanted in the left tibia of the other seven sheep. In all sheep, the right tibia was left intact. During pin implantation, the final insertion torque was measured, and a linear external fixator was mounted on the pins. Then the medial tibial mid-diaphysis was exposed and a 5-mm resection osteotomy was performed. The sheep were killed six weeks after surgery. MAIN OUTCOME MEASURES: The extraction torque was measured on four pins removed from each sheep. Radiographic pin tract rarefaction was measured on all the pins. Two pins from each sheep were used for histologic, scanning electron microscopy (SEM), and microhardness analysis. Histomorphometric analysis was carried out on the SEM specimens at x 36 magnification. RESULTS: Radiographic pin tract rarefaction was significantly lower in the hydroxyapatite coated pins than in the uncoated pins (P < 0.001). Group average insertion torque was 960 +/- 959 N/mm in the hydroxyapatite coated pins, and 709 +/- 585 N/mm in the uncoated pins (p = not significant). Group average extraction torque was 1485 +/- 1308 N/mm and 298 +/- 373 N/mm, respectively (p = 0.0001). Histomorphometric analysis showed that the group average bone-pin contact was 50.7 +/- 16.9% in the hydroxyapatite coated pins and 27.6 +/- 7.1% in the uncoated pins (p < 0.01). Microhardness analysis showed that bone tissue close to the pins was softer than bone tissue far from them. CONCLUSION: Hydroxyapatite coating is an effective method of refining the bone-pin interface and may improve the clinical results of the external fixation technique.

Animals↗

PubMed1987

The assay, purification and properties of vaccinia virus-induced uncoating protein.

Vaccinia virus cores prepared in vitro can be uncoated by incubation with extracts of cells infected with vaccinia virus, as determined by the conversion of the genome to DNase susceptibility. This uncoating activity had all the characteristics of the corresponding in vivo activity and of the agent responsible for non-genetic reactivation. Thus, it was not induced by heat-inactivated virus, nor was it produced in the presence of inhibitors of RNA or protein synthesis. The uncoating protein induced by cowpox virus will uncoat vaccinia virus cores. The uncoating protein was concentrated from infected cell extracts by ultrafiltration and purified by gel filtration and ion-exchange and affinity chromatography. It was characterized as a trypsin-like protease with a mol. wt. of 23,050. Cores treated with the purified uncoating protein had an altered sedimentation rate but no differences between treated and untreated cores were detected by electron microscopy or polyacrylamide gel electrophoresis.

Carrier Proteins↗

PubMed1990

An intragenic revertant of a poliovirus 2C mutant has an uncoating defect.

A revertant was isolated from a temperature-sensitive poliovirus 2C mutant, 2C-31, which is defective in viral RNA synthesis. This revertant, called 2C-31R1, grew well at 39 degrees C and was not defective in RNA synthesis. However, in contrast to its parental mutant, 2C-31R1 was cold sensitive and could hardly grow at all at 32 degrees C. Analysis of a single-cycle growth revealed that 2C-31R1 was defective in virion uncoating at 32 degrees C, and a substantial amount (more than 30%) of input viruses could be recovered as infectious particles from an infected cell lysate up to 6 h postinfection. The uncoating defect and the inability to grow at cold temperatures could be overcome by a brief incubation at the permissive temperature (39 degrees C) before the infection was continued at 32 degrees C. cDNA cloning and mix-and-match recombination experiments indicated that the defect in uncoating was the result of two secondary point mutations, seven nucleotides apart, in the 2C-coding sequence downstream of the inserted linker which is the original mutation in the parental 2C-31 genome. Another revertant, 2C-31R3, isolated from the same 2C-31 stock, was not defective in uncoating and appeared to be a secondary revertant that contained an intragenic suppressor for the uncoating defect. The uncoating defect of 2C-31R1 could be complemented by type 2 poliovirus. These results suggested that protein 2C, in addition to its role in viral RNA synthesis, has a function in determining virion structure.

Base Sequence↗

PubMed1990

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment.

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.

Ammonium Chloride↗

PubMed1987

Penetration and uncoating of rotaviruses in cultured cells.

Early steps of replication (penetration and uncoating) of the OSU strain of porcine rotavirus were studied in MA-104 cells. After adsorption of trypsin-treated viruses at 4 degrees, followed by a shifting of the temperature to 37 degrees, particles were seen within coated pits, coated vesicles, and secondary lysosomes, indicating that virus entry occurred by receptor-mediated endocytosis, and that uncoating (removal of the outer capsid) could occur by the effect of lysosomal enzymes. This latter aspect was studied using lysosomotropic drugs (chloroquine and ammonium chloride), which were found not to inhibit rotavirus replication, indicating that the low intralysosomal pH is not responsible for virus uncoating. The effect of Ca2+ concentration on intracellular rotavirus uncoating was investigated by treating cells with the calcium ionophore A23187, to increase the intracellular Ca2+ concentration during the early stages of virus replication. Under these conditions rotavirus uncoating did not occur, suggesting that the low Ca2+ concentration in the intracellular microenvironment may be responsible for rotavirus uncoating.

Ammonium Chloride↗

PubMed1985

Two classes of binding sites for uncoating protein in clathrin triskelions.

Clathrin released from coated vesicles or empty cages by the ATP-dependent action of uncoating protein exists as a complex with the uncoating protein. Despite its apparent consumption during a round of uncoating, we have found that uncoating protein functions as an enzyme in that it rapidly and spontaneously recycles from its product (triskelions) to its substrate (cages). The binding of uncoating protein to clathrin triskelions is a complex equilibrium that involves the interaction of uncoating protein with at least two distinct sites on the clathrin molecule. Limited proteolysis dissected clathrin into two domains, each of which contained distinct binding sites. Binding to one of these sites, located on the proximal leg of a triskelion, was dependent upon the presence of light chains and was unstable to gel filtration. Binding to the second kind of site, located on the distal portion of a triskelion leg, was stable to gel filtration and was independent of the presence of light chains.

Adenosine Triphosphatases↗

PubMed1994

ATPase activity associated with the uncoating of clathrin baskets by Hsp70.

In the presence of ATP, bovine brain hsp70 has been shown to remove clathrin from bovine brain clathrin-coated vesicles in a rapid stoichiometric initial burst followed by slow steady-state uncoating. In addition, it has been found recently that a 100-kDa cofactor is required for hsp70 to uncoat clathrin baskets prepared with the assembly protein AP-2. In this study the ATPase activity associated with uncoating was investigated, with baskets formed from clathrin and assembly proteins. Mixed assembly proteins or assembly protein AP-2 could not be used in ATPase studies because they activated the hsp70 ATPase activity even in the absence of clathrin. However, this was not the case with assembly protein AP180. A stoichiometric initial burst of ATP hydrolysis was found to accompany the initial burst of uncoating of AP180-clathrin baskets by hsp70, with 1 mol of hydrolyzed ATP/mol of released clathrin heavy chain. Furthermore, the presence of a 100-kDa cofactor was needed for both processes. These results suggest that an initial burst of uncoating occurs with all clathrin baskets, that an initial burst of ATP hydrolysis accompanies this initial burst of uncoating, and that a 100-kDa cofactor is required for both.

Adenosine Triphosphatases↗

PubMed1983

The retention mechanics of LTI carbon, carbon-coated aluminum oxide, and uncoated aluminum oxide dental implants.

The in vitro mechanical behavior of identical geometry LTI carbon, carbon-coated aluminum oxide, and uncoated aluminum oxide blade-type dental implants has been evaluated using rosette type strain gauges and a LVDT system. The implants served as a distal abutment for a three-unit fixed prosthesis and functioned for a period of 2 years in female baboons. The comparison of the LTI carbon implants to the carbon-coated aluminum oxide implants allowed for a study of the effect of implant elastic modulus on the mechanical behavior, while the comparison of the carbon-coated and uncoated aluminum oxide implants allowed for a study of the effect of chemical composition at the tissue-implant interface. The results of the mechanical testing indicate that the implant displacement response of the LTI carbon implants was greater than that of the carbon-coated and uncoated aluminum oxide implants. Little difference in displacement response was observed for the carbon-coated and uncoated aluminum oxide implants. No clear trend in the strain response of the buccal mandibular bone was observed for the three implant systems. The greatest differences in strain response, however, was observed between the uncoated aluminum oxide implants and the LTI carbon and carbon-coated aluminum oxide implants.

Aluminum Oxide↗

PubMed1985

Suppression of Trypanosoma congolense, T. vivax and T. brucei infection rates in tsetse flies maintained on goats immunized with uncoated forms of trypanosomes grown in vitro.

Significant suppression in the incidence of cyclical development of Trypanosoma congolense, T. vivax and T. brucei occurred in Glossina morsitans centralis maintained on goats immunized with in vitro-propagated uncoated forms of T. congolense, T. vivax and T. brucei, respectively. This was observed when tsetse given a T. congolense-infected feed were subsequently maintained on uninfected immunized goats and also when uninfected tsetse were fed on immunized goats infected with T. congolense, T. vivax and T. brucei. Suppression of infection rates in tsetse was trypanosome species specific, but was independent of the trypanosome stock used for immunization of goats. These findings were reflected in antibody responses to uncoated trypanosomes, as measured by immunofluorescence and the solid-phase immunoradiometric binding assay. Thus, antibody from goats immunized with uncoated trypanosomes of one species exhibited minimal reactivity with uncoated forms of other species of trypanosomes, but showed high levels of activity with uncoated forms of the same or unrelated stocks of the same species. However, in view of the range of hosts upon which tsetse feed, it is open to question whether the use of a vaccine which suppresses trypanosome infection rates in tsetse would have any significant effect in the field.

Animals↗

PubMed1996

Bidirectional uncoating of the genomic RNA of a helical virus.

An essential step in the initiation of a virus infection is the release of the viral genome from the other constituents of the virus particle, a process referred to as uncoating. We have used reverse transcription and polymerase chain reaction amplification procedures to determine the rate and direction of in vivo uncoating of the rod-shaped tobacco mosaic virus. The virus particles contain a single 6.4-kb RNA molecule that lies between successive turns of a helical arrangement of coat protein subunits. When the particles are introduced into plant cells, the subunits are removed via a bidirectional uncoating mechanism. Within 2-3 min, the part of the viral RNA from the 5' end to a position >70% toward the 3' end has been freed of coat protein subunits. This is followed by removal of subunits from the 3' end of the RNA and sequential uncoating of the RNA in a 3'-to-5' direction. An internal region of the viral RNA is the final part to be uncoated. Progeny virus particles are detected in the cells 35-40 min after inoculation.

Base Sequence↗

PubMed2000

Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells.

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.

Adaptor Proteins, Vesicular Transport↗