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In a virus particle, the genome is highly condensed and protected by proteins and membrane bilayers. Before it can be replicated in a new host cell, uncoating must take place. Recent studies on enveloped and nonenveloped animal viruses indicate that uncoating occurs through complex, multistep processes triggered by virus-host-cell interactions.
The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose. The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential. In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant. Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction. This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process.
Determination of infectious progeny virus and in vivo labelling with [(35)S]methionine followed by immunoprecipitation demonstrates that the major receptor group human rhinovirus HRV14 is able to infect HeLa cells in the presence of the V-ATPase inhibitor bafilomycin A1. However, host cell shut off is delayed and viral yield is decreased in the presence of the drug. Uncoating can thus take place under conditions that prevent endosomal acidification indicating that it is catalysed by the viral receptor alone. Since transport is arrested in early endosomes upon inhibition of vesicle acidification, the data also suggest that productive uncoating takes place from early endocytic compartments.
Rapid (< 5 min) high-performance isoelectric focusing was performed in uncoated fused-silica capillaries to resolve isoforms of monoclonal antibodies and to determine their isoelectric points (pI). The methodology involved the use of a 32 cm (effective length 9 cm) x 50 microns I.D. uncoated capillary. (Hydroxypropyl)methyl cellulose was used as an additive to suppress analyte-wall interaction and to precisely control electroosmotic flow so that focusing and mobilization of focused zones past detector occur simultaneously. Urea was included in the separation medium to solubilize the antibodies that precipitated at their point of focusing. The methods with and without urea used ampholytes pH 5-8 to generate a demonstrate linear gradient between pH 5.4 and pH 7.2, based on the separation of various protein standards. Reproducibility [< 2% (R.S.D.)] of the migration times (corresponding to the detectable isoforms of the antibodies) was obtained by using two sets of reagents and capillaries on three consecutive days. pI values determined from day-to-day with a reference standard were shown to vary by only 0.01 pH unit. The described capillary isoelectric focusing methods provided a rapid, simple and reproducible way of monitoring micro-heterogeneity and pI of the murine monoclonal antibodies investigated.
Rotaviruses are now regarded as important causes of diarrhoea in man, cattle, pigs, mice, and possibly other animals. Characteristically, disease occurs in newborn and young animals, and infection seems limited to the differentiated gut epithelial cells. The major surface polypeptide of the calf scours rotavirus is glycosylated, and highly purified beta-galactosidase (lactase) interacts with the virus in vitro causing removal of the outer shell of the capsid (uncoating). It is suggested that lactase present in the brush border of the intestinal epithelial cell performs a similar function in vivo by acting as a combined receptor and uncoating enzyme for the rotavirus. This hypothesis is consistent with the observations that rotaviruses seem to infect only gut epithelial cells, and that infant animals, whose lactase concentrations are generally higher than those of adult animals, seem more susceptible to rotavirus infections. Implications of the hypothesis include possible new approaches to laboratory cultivation of rotaviruses, which should be more successful in cells selected for surface lactase activity, and the suggestion that the epidemiology of human rotavirus infections may be influenced by the fact that different ethnic groups have different lactase levels (and hence lactose intolerance) in adulthood.
The lung concentration of coated and uncoated mineral fibres of more than 10 micron in length was determined by light microscopy in 57 autopsied subjects (23 out of which professionally exposed to asbestos) with pleural plaques but no other asbestos-related diseases. Comparisons were made with 57 controls matched for sex, age and smoking habits, who did not show any asbestos-associated diseases or history of previous occupational exposure. Subjects with pleural plaques had a median concentration of coated fibres consistently greater than that observed in those without. However, no substantial differences were found between the two studied groups when uncoated and total fibres were considered. The present study suggests that the factors related to the fibre coating and to the pleural plaque formation may be indirectly connected, but not necessarily restricted to the number and nature of the fibres, or to the professional or environmental conditions of exposure.
The tristimulus color coordinates CIELAB and associated parameter Color Intensity (CI) have been shown to be a quantifiable variable for whiteness of uncoated tablets. Whereas any of L*, a* or b* indicates the discoloration of white tablets to a certain degree, it alone cannot reflect the full extent of discoloration. The CI has been defined which is able to describe the discoloration kinetics with acceptable regression coefficients. The evaluation of the CIELAB values from the stability data has shown that the discoloration of the white tablets means an intensification of yellowish or brownish color which is manifested by more or less constant hue angle (hab) values and increasing chroma (Cab*) values. In the view of these data the discoloration kinetics can physically be expressed by the CI. With the CI values the discoloration kinetics can be calculated by linear or polynomial regression with acceptable confidence intervals. The discoloration rates determined under several storage temperatures follow the Arrhenius equation and the activation energy can be estimated for the products. The CI values are unambiguously connected to the visual perception of the corresponding tablets. By means of the discoloration kinetics based on the CI values, it has become possible to statistically determine, the period of time uncoated tablets remain white.
Pellets of different shape, varying from spherical to cylindrical, without and with film coating were tested for their drug release properties. For non-disintegrating uncoated pellets, drug release was found to be inversely related to the pellet porosity. A change of 5% in porosity doubled the value of the mean dissolution time (MDT). As coat thickness increased, the MDT value of coated pellets increased. For those pellets, which are nearly spherical, once a thickness of about 20 microm had been achieved, there was little further reduction in retardation. Pellets produced by extrusion/spheronisation appeared to prolong drug release to a larger extent than those where the extrusion step had been omitted. There was a strong inverse relationship between the surface area by volume of the coated pellets and the value of the MDT. The values of the relative dispersion coefficient (RD), which is an indicator of the drug release mechanism, were related to the amount of fluid used to manufacture the pellets and the pellet shape, in a similar fashion for both uncoated and coated pellets. This suggested that the presence of the film coating changed the rate but not the mechanism of drug release.
The large GTPase dynamin is required for budding of clathrin-coated vesicles from the plasma membrane, after which the clathrin coat is removed by the chaperone Hsc70 and its cochaperone auxilin. Recent evidence suggests that the GTP-bound form of dynamin may recruit factors that execute the fission reaction. Here, we show that dynamin:GTP binds to Hsc70 and auxilin. We mapped two domains within auxilin that interact with dynamin, and these domains inhibit endocytosis when overexpressed in HeLa cells or when added in a permeable cell assay. The inhibition is not due to impairment of clathrin uncoating or to altered clathrin distribution in cells. Thus, in addition to its requirement for clathrin uncoating, our results show that auxilin also acts during the early steps of clathrin-coated vesicle formation. The data suggest that dynamin regulates the action of molecular chaperones in vesicle budding during endocytosis.
PURPOSE: The purpose of this research is to obtain the tablet coating distribution from weight distributions of uncoated and coated tablets. METHODS: The method of deconvolution with digital smoothing was used to calculate the distribution of coating applied to a tablet population from separate random measurements of individual uncoated and coated tablets. RESULTS: It was demonstrated that the calculated coating weight distribution agrees well with the measured distribution. The effect of the smoothing factor on the solution is illustrated. CONCLUSIONS: This method can be used during development to facilitate process scale-up/optimization. In routine production, the method can assess the reproducibility and consistency of a coating process.
This study was undertaken to evaluate whether the pyrolytic carbon coating of polyethylene terephthalate induces complement activation. Complement activation induced by pyrolytic carbon-coated polyethylene terephthalate (PET+PC) in comparison with uncoated polyethylene terephthalate (PET) was assessed on whole blood collected with heparin. The activation of the classic pathway was evaluated by C4d fragment enzyme immunoassay. The activation of the alternative pathway was evaluated with Bb fragment enzyme immunoassay. The results show that uncoated PET activates the alternative pathway, but not the classic one. PET+PC does not induce complement activation, not even through the alternative pathway. Pyrolytic carbon coating therefore contributes to improving blood compatibility.
Hydroxyapatite-coated and uncoated titanium screws were inserted in the rabbit tibial metaphysis and evaluated by histomorphometry after 6 months of follow-up. There was no difference in direct bone contact between the HA-coated implants and the uncoated controls. The effect of HA-coating on screw-shaped implants seems to be uncertain, in contrast to unthreaded cylindrical designs, where there is more abundant documentation in favour of HA-coated implants.
Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1 +/- 0.1 microM and 1.4 +/- 0.7 mM respectively at 25 degrees C. This corresponds to delta G0' = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, delta H0' was calculated to -36 +/- 2 kJ/mol. This gives delta S0' = 6.7 J/mol per K. Adenosine 5'-[gamma-thio]triphosphate, ADP, adenosine 5'-[beta, gamma-imino]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 microM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.
AIMS: Heparin coating of stents is thought to reduce stent thrombosis and restenosis rates. However, clinical data comparing coated and uncoated stents of the same model are lacking. We compared the heparin coated (C) and the uncoated (U) version of the Jostent stent with regard to the clinical and angiographic outcome after 6 months. METHODS AND RESULTS: Provisional stenting was done in 277 patients and 306 lesions; only 40 were Benestent-II like lesions. Delivery success rate was 98.4%. Both groups (C/U: n=156/150 lesions) were comparable in clinical and procedural data. Post stenting, reference diameter (C/U: 2.68+/-0.56/2.66+/-0.53 mm) and minimal lumen diameter did not differ (C/U: 2.48+/-0.47/2.48+/-0.52 mm). During follow-up the rate of subacute stent thrombosis (C/U: 1.9%/1.3%) and myocardial infarction did not differ. Angiography at the 6-month follow-up (79.4%) revealed no difference in restenosis rate (C/U: 33.1%/30.3%). Risk factors for restenosis were a type B2/C lesion (P<0.02), a stented segment longer than 16 mm (P<0.006) and a stent inflation pressure <14 bar (P<0.0063). CONCLUSION: Corline heparin coating of the Jostent has no impact on the in-hospital complication rate, stent thrombosis or restenosis. The Jostent design gives a high procedural success rate and satisfying result at 6 months in an everyday patient population undergoing provisional stenting.
OBJECTIVE: To determine if the Trillium Biopassive Surface (Medtronic Cardiopulmonary, Minneapolis, MN) coating added to the cardiopulmonary bypass oxygenator reduces inflammatory mediators, blood loss, and transfusion requirements. DESIGN: Prospective, randomized, and blinded human trial. SETTING: Tertiary care academic medical center. PARTICIPANTS: Thirty adult patients undergoing elective coronary artery bypass graft surgery. INTERVENTIONS: Patients received visually identical coated or uncoated oxygenators. MEASUREMENTS AND MAIN RESULTS: Hemoglobin, hematocrit, leukocyte count, platelet count, terminal complement complex, complement activation, myeloperoxidase, beta-thromboglobulin, prothrombin fragment 1.2, plasmin-antiplasmin, heparin concentration, activated coagulation time, and fibrinogen concentration were measured. Blood loss and blood product usage were recorded. In both groups, there were significant inflammatory alterations with the initiation of cardiopulmonary bypass. In the postprotamine samples, the coated oxygenator group had small but significant increases in hemoglobin, hematocrit, and leukocyte count. There were no differences in inflammatory mediators, blood loss, or transfusion requirements between the coated and uncoated groups. CONCLUSION: This human trial of Trillium Biopassive Surface-coated oxygenators did not show clinical benefits or clinically important biochemical results.
A total of 70 patients undergoing replacement of the aortic bifurcation between October 1983 and June 1985, were studied 5 years postoperatively for graft patency. In 35 cases a collagen-coated knitted Dacron prosthesis (group I) was implanted, while an identical, but uncoated, Dacron graft was used in a second group of 35 patients (group II). Distribution of vascular risk factors were similar in both patient groups. The underlying disease was aortoiliac occlusive disease in 24 patients of group I and in 33 of group II. Aortoiliac aneurysmal disease was present in 10 patients of group I and in 2 patients of group II. One patient of group I was suffering from Takayasu's disease of the descending type. At follow-up, all grafts were patent in group I, while in group II 8 distal limb occlusions occurred between 6 and 46 months postoperatively. This represents a 5-year limb patency rate of 100% for collagen-presealed grafts versus 88.6% for uncoated prostheses (p less than 0.01). A higher incidence of distal anastomotic aneurysms was found in group I (7/70 limbs) compared to group II (1/70 limbs) (p less than 0.05). It is concluded that long-term patency of knitted Dacron prostheses in aortic bifurcation replacement is not reduced by presealing with collagen. It appears that this type of coating may actually enhance long-term patency after reconstruction of the aortoiliac axis. The higher rate of distal anastomotic aneurysm formation in patients with presealed grafts, however, could not be correlated with a failure of the sealant material or with the underlying disease of the patients involved.
The authors studied the ingrowth dynamics of bone tissue into the pores of Hydroxyapatite-coated (plasma-spraying technique) and uncoated wiremeshs of pure titanium in an infected implantation site. Samples of the tested materials were implanted into the femora of 15 adult Göttingen minipigs. Just before implantation they were contaminated with Staphylococcus aureus. The animals were sacrificed after 4, 8, 12 and 24 weeks. Undecalcified ground-sections of bone tissue were prepared (app. 100 microns) and stained with toluidine blue for comparative histological evaluation. The HA-coated implants demonstrated advanced new bone formation already after 4 weeks and nearly complete osseointegration after 12 weeks although all samples showed gross and histological signs of persisting infection. Comparable reactions of the uncoated implants could only be observed after 24 weeks. Signs of degradation of the Hydroxyapatite-coating could be seen in contact to soft tissue and were more extensive compared to the behaviour in an uninfected site. The results and possible clinical consequences are discussed.
The molecular events underlying the immediate steps of retroviral uncoating, occurring after membrane fusion and leading to the formation of an active reverse transcription complex, are not known. To better understand these processes, we have developed a cell-free system that recapitulates these early steps of retroviral replication by using avian sarcoma and leukosis virus as a model retrovirus. The substrates used in this system are viral particles that are trapped before completing membrane fusion. These virions are induced to fuse out of endosomes and the viral cores are released into solution where they are amenable to biochemical manipulation. This system revealed that membrane fusion is not sufficient to stimulate the formation of a reverse transcription complex. Instead, ATP hydrolysis and cellular factors >5 kDa in size are required. Furthermore, later steps of avian sarcoma and leukosis virus reverse transcription were stimulated by nuclear factors. The cell-free system should now allow for the definition of retroviral uncoating mechanisms and facilitate the identification and characterization of the cellular factors involved.