Furan-Based Porphyrins: Tetraoxa
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Journal Article↗
Biomedical subjects
T Berg
Publications and source records attributed to T Berg.
Antiviral combination therapy for lamivudine-resistant hepatitis B reinfection after liver transplantation.
Development of resistance is a major issue in antiviral treatment of hepatitis B reinfection after liver transplantation. Antiviral combination therapy is discussed for therapy or prevention of this breakthrough of viral replication. Eight patients were enrolled into this retrospective analysis after liver transplantation for chronic hepatitis B infection. All had reinfection of the graft and breakthrough of HBV during consecutive famciclovir and lamivudine monotherapy. Subsequently a combination therapy with lamivudine and interferon-alpha 2a (group I, n = 4) or lamivudine and famciclovir (group II, n = 4) was initiated. Combination therapy was started 61 months (group I) and 25 months (group II) after liver transplantation. It markedly reduced the viral replication rate in all patients despite lamivudine resistance. In group I three of four patients and in group II two of four patients became HBV-DNA negative. Two long-term responders were observed in group I, and none in group II. No patient became HBsAg negative or lost HbeAg. Pretreatment elevated ALT and AST levels were significantly reduced. No severe complications, and especially no rejection episodes, occurred. Lamivudine in combination with other antiviral agents, especially interferon-alpha, might be a therapeutic option for hepatitis B reinfection after liver transplantation. Suppression of virus replication to the point of undetectable values is possible even in patients with lamivudine-resistant virus mutations.
2-Aminopurine↗
Lamivudine as first- and second-line treatment of hepatitis B infection after liver transplantation.
Lamivudine and famciclovir have expanded therapeutical options for HBV infection after liver transplantation. First studies confirm good antiviral effects of both, but at present the major problem seems to be a rapid resistance formation in immunosuppressed patients. Thirty-four adult patients with HBV recurrence despite passive immunoprophylaxis and seven with de novo infection after orthotopic liver transplantation (OLT) were treated with 100-150 mg lamivudine daily. Patients were either treated directly after infection (n = 14) or after breakthrough of viral replication during an initial famciclovir therapy (n = 27). All patients except two responded to treatment with a reduction of serum HBV-DNA of over 50%. Thirty-one patients (76%) turned HBV-DNA-negative during lamivudine therapy. Viral breakthrough was observed in 14 of these patients after 4-13 months of treatment. A total of 17 patients (40%) remained HBV-DNA-negative for more than 12 months. Only nine patients eliminated HBsAg, of which four had and an HDV coinfection. None of the HBeAg-positive patients converted to anti-HBe. Most patients showed a prompt and significant reduction of aspartate aminotransferase (ALAT) levels. No severe complications occurred. Therefore, a safe and effective therapy of HBV infection after transplantation is possible with lamivudine. Viral replication is suppressed even in patients who revealed breakthrough during famciclovir therapy. Resistance formation as a major drawback occurred in one third of the patients within the first year of treatment.
2-Aminopurine↗
Phagocytosis and chemiluminescence response of granulocytes to monodisperse latex particles of varying sizes and surface coats.
The response of human granulocytes to polystyrene latex beads of diameter 0.1-7 microm was measured by luminol-dependent chemiluminescence. In all instances, the response to beads of 3-7 microm was definitely higher than with smaller beads. In protein-free medium, the chemiluminescence response was slow compared to that of opsonized zymosan, and the highest response was only 9% of the response to opsonized zymosan. Scanning electron microscopy showed that granulocytes in suspension bound the particles, occasionally by extending rope-like protrusions. When the beads were coated with albumin, the chemiluminescence diminished to about 1/3 of that seen with uncoated beads; however, preincubating the beads in serum led to a large increase with beads of 1.1 microm (to 25% of the maximal response to opsonized zymosan) and 3.19 microm (to 42%), but with the smallest beads, no increase was noted. "Priming" of the cells with tumor necrosis factor-alpha caused a further increase with serum-coated beads. When uncoated beads of 1.1 microm were tested with "primed" cells, there was an increase of 6 times in the chemiluminescence compared to un-"primed" cells.
Blood↗
Efficacy of a short-term ribavirin plus interferon alpha combination therapy followed by interferon alpha alone in previously untreated patients with chronic hepatitis C: a randomized multicenter trial.
BACKGROUND: Combination therapy with interferon alpha (IFNalpha) plus ribavirin has been shown to improve the sustained response rate in patients with chronic hepatitis C but there is little information regarding the lengths of time for this therapeutic regimen. In this study we therefore tried to evaluate whether the analysis of different virological parameters could provide new clues with respect to the early determination of the efficacy of this form of combination therapy. Furthermore, we also examined whether short-term induction combination therapy followed by IFNalpha alone is more effective than monotherapy in mounting an initial as well as a sustained virological response. METHODS: 185 patients with histologically proven chronic hepatitis C (mean age 42 years (range 19-65 years); 110 males, 75 females) were enrolled in the study. The patients were randomly assigned to receive, over the first 12 weeks, either interferon alpha 2a 6 million units (MU) three times weekly plus ribavirin 14 mg/kg per day (n=93) or the same dose of IFNalpha alone (n=92). Patients with a virological response (serum HCV RNA undetectable) after 12 weeks were subsequently treated with 3 MU IFNalpha alone thrice weekly for a further 40 weeks. Otherwise, treatment was discontinued. After the end of treatment, patients were followed up for 24 weeks. RESULTS: Patient characteristics at baseline were not significantly different in the two treatment groups. An initial virological response at week 12 was seen in 61 (66%) patients receiving IFNalpha plus ribavirin and in 44 (48%) being treated with IFNalpha alone (p=0.015) and this improvement in the response rate was mainly restricted to HCV genotype 1-infected patients (58% vs. 38%). In contrast, end-of-treatment (week 52) and sustained virological response rates were similar in both groups (37% vs. 29% and 26% vs. 17% [p=0.1], respectively). Interestingly, patients with HCV genotype 3, however, clearly benefited from short-term combination therapy. Thus, sustained virological response rates in these patients significantly increased from 25% (IFNalpha monotherapy) to 59% (combination therapy) (p=0.05). CONCLUSIONS: Short-term combined therapy for 12 weeks is more effective than the monotherapy with respect to the induction of an initial virological response but this effect applies only to genotype 1-infected patients. However, there is no significant difference between both therapeutic schedules with regard to the induction of sustained response. Although HCV genotype 3-infected patients seem to benefit from this short-term combined therapy, prolonged combined therapy may be necessary in HCV genotype 1-infected patients.
Adult↗
Randomized, double-blind, placebo-controlled trial of interferon alfa2a with and without amantadine as initial treatment for chronic hepatitis C.
Although the antiviral effects of amantadine sulphate (1-aminoadamantan sulphate) have not been characterized for the hepatitis C virus (HCV), previous pilot studies have suggested promising results in patients with chronic hepatitis C. The aim of the present study was to compare the efficacy, safety, and health-related quality of life (HRQOL) of interferon alfa (IFN-alpha) alone or in combination with oral amantadine for treatment of chronic hepatitis C. One hundred nineteen previously untreated patients with chronic hepatitis C were randomly allocated to treatment with IFN-alpha2a at a dose of 6 megaunits 3 times a week subcutaneously for 24 weeks, followed by 3 megaunits thrice weekly for an additional 24 weeks plus amantadine sulphate administered orally 100 mg twice a day for 48 weeks or the same IFN regimen plus a matched placebo. The primary endpoint was undectable serum HCV RNA (<1,000 copies/mL) at week 24 after treatment. At the end of treatment and the 24-week follow-up period serum HCV RNA was undetectable in 20 (34%) and 6 (10%) of the 59 patients treated with the combination IFN-alpha plus amantadine and in 20 (33%) and 13 (22%) of the 60 patients treated with IFN-alpha alone, respectively (P = n.s.). Discontinuation of therapy for adverse events was similar in both treatment groups. Although treatment with IFN-alpha worsened HRQOL, combination with amantadine showed a substantial trend to improve fatigue and vigor. In conclusion, combination therapy IFN-alpha plus amantadine is as effective as IFN-alpha monotherapy in previously untreated patients with chronic hepatitis C.
Adolescent↗
Mutations in the E2-PePHD and NS5A region of hepatitis C virus type 1 and the dynamics of hepatitis C viremia decline during interferon alfa treatment.
Both a double-stranded RNA-dependent protein kinase (PKR)-phosphorylation homology domain (PePHD) within the E2 protein and a PKR-binding domain within the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 isolates inhibit the function of the interferon alfa (IFN-alpha)-induced antiviral effector protein PKR in vitro. We investigated whether the mutational pattern of the E2 region (codons 618-681, including PePHD) of 81 HCV genotype 1-infected patients (HCV-1b [n = 54], HCV-1a [n = 27]) influences the response to IFN-alpha. Initial viral decline (DeltaHCV RNA) was determined at week 1 hereby covering the effector reactions of IFN-alpha-mediated first phase and the immune-mediated second phase. DeltaHCV RNA less than 50% (group 1); DeltaHCV RNA greater than 50% but less than 90% (group 2); and DeltaHCV RNA > or =90% (group 3) were differentiated. The PePHD region was highly conserved; the few mutations (5 patients) did not correlate with DeltaHCV RNA or sustained virologic response to IFN-alpha. Within the flanking regions before and after PePHD (codons 618-681) 72 of 81 patients (89%) had 2.6+/-0.17 mutations (median, 3; range, 1-8) that did not correlate with treatment response. Sequence analysis of the NS5A protein (codons 2,209-2,274, including interferon sensitivity determining region [ISDR]) in 39 of 81 patients showed a higher mean number of mutations in the ISDR (codons 2,209-2,248) in groups 2 (1.28+/- 0.43 [n = 18]) and 3 (1.89+/-0.54 [n = 9]) than in group 1 (0.67+/- 0.19 [n = 12]; P =.049 group 1 vs. 3) and a mutant type ISDR (e.g., > or =4 mutations) was significantly more frequent in sustained virologic responders than in nonresponders or relapsers (2 of 4 [50%] vs. 2 of 35 [6%]; P =.045). Thus, NS5A appears to be functionally relevant in IFN-alpha-induced effector reactions.
Amino Acid Sequence↗
[Effect of ribavirin on dynamics of hepatitis C viremia in interferon alpha-treated patiens with response or no response].
Combination therapy with interferon-alpha (IFN alpha) plus Ribavirin has been shown to improve the response rate in patients with chronic hepatitis C as compared to IFN alpha alone. However, the mode of anti-viral action of Ribavirin is still unknown. To prove, whether Ribavirin has any additional effect on the decline of hepatitis C viremia during the first weeks of treatment patients with and without combination therapy were compared. Kinetic studies were performed in patients who either responded to IFN alpha alone or IFN alpha plus Ribavirin combination as well as in nonresponders to both forms of therapeutic approaches. 64 IFN alpha naive patients with histologically proven chronic hepatitis C were included in the study. Patients were randomized to receive either IFN alpha-2a (Hoffmann-La Roche) 6 MU thrice weekly or IFN alpha 6 MU tiw plus Ribavirin (Meduna) 14 mg/kg/day for 12 weeks. 37 patients (58%) became HCV RNA-negative (= responders; 17 [46%] with IFN alpha alone, and 20 [54%] with combination therapy). 27 patients remained HCV RNA-positive (= non-responders; 13 [48%] with IFN alpha alone, and 14 [52%] with combination therapy). HCV RNA concentrations were measured in all patients at baseline as well as 1, 2, 4, and 12 weeks after the start of treatment (bDNA assay, Chiron). Using nonradioactive single-stranded conformation (SSCP)-analysis of the HCV hypervariable region 1 we investigated further whether initial viral decline is correlated with changes in viral quasispecies distribution. In primary responders, ribavirin did not influence hepatitis C viremia decline which was of biphasic nature. Also in nonresponders HCV RNA levels decreased after one week of treatment irrespectively of the mode of therapy (mean 10.0 +/- 2.3 to 5.5 +/- 1.1) (phase 1). In the following weeks, however, 2 types of HCV dynamics could be observed (phase 2). In patients with combination therapy, a further reduction of viremia level could be observed, whereas viremia levels in patients with IFN alpha alone slightly increased (week 12: 3.0 +/- 0.5 MEq/mL [combination, n = 15] vs. 7.5 +/- 2.9 MEq/mL [IFN alpha-mono, n = 12]). The individual response of these nonresponder patients showed, however, marked differences (range percentage decline after 4 weeks, 0-98%). Changes in the viral population (quasispecies distribution) as cause of these differences could be excluded by SSCP-analysis of PCR products of the HCV hypervariable region 1. Ribavirin in combination with IFN alpha exerts an additional anti-viral/immunmodulatory effect which manifests itself in phase 2 of hepatitis C viremia decline. The biphasic decline of hepatitis C viremia also observed in IFN alpha-nonresponders can not be explained by the selection of primary IFN alpha-resistant viral variants. The individual differences in the dynamic of hepatitis C viremia observed in the so called "nonresponders" imply that the term "nonresponder" should be redefined, considering our observation that a marked viral decline can occur in these patients.
Adult↗
[Therapy of recurrent hepatitis B infection after liver transplantation. A retrospective analysis of 200 liver transplantations based on hepatitis B associated liver diseases].
BACKGROUND: Before introduction of passive immunoprophylaxis and new antiviral nucleoside analogues the course of hepatitis B recurrence after liver transplantation could hardly be influenced. The result was a inferior graft survival. In the present retrospective analysis of the efficacy of hepatitis B therapy after liver transplantation was analysed retrospectively. PATIENTS AND METHODS: Between 1988 and 1998 in total 179 patients were transplanted due to hepatitis B related liver failure at our centre. All patients received passive immunoprophylaxis with hepatitis B immunoglobulin. In case of reinfection after 1993 an antiviral therapy with famciclovir 1500 mg daily was initiated (n = 26), since 1996 lamivudine (100-150 mg daily) was used (n = 12). In case of viral breakthrough under famciclovir treatment or prophylaxis therapy was switched to lamivudine (n = 22). In case of ineffectiveness of lamivudine an antiviral combination therapy with lamivudine and interferon (n = 4) or lamivudine and famciclovir (n = 4) was initiated. Before availability of antiviral agents or in case of viral breakthrough in total 12 patients were retransplanted due to acute or chronic reinfection. RESULTS: With passive immunoprophylaxis reinfection rate was 33%, 43% and 44% after 1, 3 and 5 years respectively. Without antiviral treatment 52% of patients died within the first year after reinfection. Antiviral therapy with lamivudine or famciclovir improved the one year survival after reinfection to 79%. Suppression of viral replication was more effective with lamivudine. Under lamivudine 26 patients (76%) became HBV-DNA negative, 9 patients HBsAg negative (26%). In contrast no patient became HBsAg negative during famciclovir therapy. Lamivudine was effective also after famciclovir breakthrough in 94% of patients. In case of lamivudine resistant reinfection viral replication could be suppressed with an antiviral combination therapy up to negative HBV-DNA in the hybridization assay. Severe side effects were not observed during any of the antiviral therapies. The graft survival after retransplantation for hepatitis B reinfection was 42% and 25% after one and 3 years. CONCLUSION: Whereas it is generally accepted, that passive immunoprophylaxis lowers the reinfection rate it could be shown in the present study, that antiviral treatment lowers mortality of hepatitis B reinfection. The major problem of lamivudine and famciclovir is viral resistance formation. In this case an antiviral combination therapy might be useful, whereas retransplantation for hepatitis B reinfection should be considered carefully due to inferior graft survival rates.
2-Aminopurine↗
The release of nickel and other trace elements from electric kettles and coffee machines.
The release of nickel, chromium and lead from electric kettles to water under conditions simulating regular household use was investigated. Ten out of 26 kettles sold on the Danish market released more than 50 micrograms/l nickel to water, whereas neither lead nor chromium was released in any significant amount. Fifty micrograms/l of nickel in water was chosen as the threshold of action, because concentrations below this value were considered unlikely to provide outbreaks of eczema for those consumers suffering from contact allergy to nickel, who are also sensitive to the content of nickel in the diet. This first part of the study was followed up by a dialogue between the kettle producers and the Danish authorities, leading to a change of construction or design for those kettles that did not comply with the criteria. As a follow-up study another ten kettles were studied to check whether compliance was improved. Two of these ten kettles still released more than 50 micrograms/l nickel to water under the test conditions. These two kettles, however, were subsequently withdrawn from the market. Coffee machines tested similarly did not release aluminium, lead, chromium or nickel in quantities of any significance.
Chromium↗
Transjugular intrahepatic portosystemic shunt. Experiences at a liver transplantation center.
OBJECTIVE: Transjugular intrahepatic portosystemic shunt (TIPS) placement is an established therapy for portal hypertension that leads to variceal bleeding or refractory ascites. We present experiences of the role of TIPS at a liver transplantation center. MATERIAL AND METHODS: One hundred and ten patients were referred to the Radiological Department for TIPS placement. One of the 110 patients had recurrent cirrhosis after liver transplantation with refractory ascites. Function of the TIPS was controlled with Doppler US at 1 day, 1 week, 4 weeks and 4 months after TIPS placement and subsequently every 3 months. Shunt insufficiency was supposed when the blood flow velocity within the stent tract was under 50 cm/s and was an indication for TIPS revision. RESULTS: TIPS was placed in 101 patients. After TIPS placement, 10 patients underwent liver transplantation. While waiting for the new liver, none of them developed variceal rebleeding, ascites or other complications of portal hypertension. Two of the 101 patients had episodes of rebleeding. The frequency of patients undergoing TIPS revision within the first year after TIPS placement was 67.5%, within the second year 38.0% and within the third year 24.4%. The revisions led to sufficient reduction of the portosystemic pressure gradient. CONCLUSION: In some liver transplant candidates, TIPS can be useful in minimizing the risk of complications of portal hypertension during the waiting time for a liver transplantation. TIPS can be monitored by Doppler US and revised if occlusion occurs.
Adult↗
Mutations in the protein kinase-binding domain of the NS5A protein in patients infected with hepatitis C virus type 1a are associated with treatment response.
An interaction of the hepatitis C virus (HCV) NS5A protein with the interferon (IFN)-alpha-inducible double-stranded RNA-activated protein kinase (PKR) was demonstrated in vitro. The clinical correlation between amino acid mutations within the HCV NS5A region and response to antiviral treatment is controversial. Thirty-two patients chronically infected with HCV-1a, who were treated with IFN-alpha with or without ribavirin, were studied. The carboxy-terminal half of HCV NS5A was sequenced and was investigated by phylogenetic and conformational analyses. Eight patients achieved a sustained virologic response. An end-of-treatment response but relapse thereafter was observed among 8 patients, whereas 16 patients were nonresponders. The median number of mutations within the PKR-binding domain but not within the previously described IFN sensitivity-determining region was significantly higher for patients with sustained (3 mutations [range, 1-5]) or end-of-treatment (4 mutations [range, 1-5]) virologic response than for nonresponders (2 mutations [range, 0-3]) (P=.0087). Phylogenetic and conformational analyses of NS5A sequences allowed no differentiation between sensitive and resistant strains.
Adult↗
Replication of staphylococcal multiresistance plasmids.
Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.
Amino Acid Sequence↗
Retreatment with interferon-alpha and ribavirin in primary interferon-alpha non-responders with chronic hepatitis C.
BACKGROUND/AIMS: Combination therapy with interferon-alpha (IFN-alpha) plus ribavirin is more efficacious than IFN-alpha monotherapy in previously untreated patients with chronic hepatitis C and patients with IFN-alpha relapse. Only limited data are available in IFN-alpha non-responders. In a multicenter trial we therefore evaluated the efficacy of combination therapy in IFN-alpha-resistant chronic hepatitis C. METHODS: Eighty-two patients (mean age 46.8 years, 54 males, 28 females) with chronic hepatitis C were treated with IFN-alpha-2a (3 x 6 MIU/week) and ribavirin (14 mg/kg daily) for 12 weeks. Thereafter, treatment was continued only in virological responders (undetectable serum HCV RNA at week 12) with an IFN-alpha dose of 3 x 3 MIU/week and without ribavirin for a further 9 months. The primary study endpoint was an undetectable HCV RNA by RT-PCR at the end of the 24-week follow-up period. RESULTS: After 12 weeks of combination therapy, an initial virological response was observed in 29 of 82 (35.4%) patients. Due to a high breakthrough rate after IFN-alpha dose reduction and ribavirin discontinuation, an end-of-treatment response was only achieved in 12 of 82 (14.6%) patients. After the follow-up period, a sustained virological response was observed in 8 of 82 (9.8%) patients. Infection with HCV genotype 3 was the only pretreatment parameter, which could predict a sustained response (HCV-1, 5%; HCV-3, 57.1%; p < 0.001). CONCLUSIONS: Despite a high initial response rate of 35.4%, sustained viral clearance was achieved only in 9.8% of the retreated primary IFN-alpha non-responders. Higher IFN-alpha induction and maintenance dose, as well as prolonged ribavirin treatment may possibly increase the virological response rates in non-responders, particularly in those infected by HCV-1.
Adolescent↗
Fc receptor mediated endocytosis of small soluble immunoglobulin G immune complexes in Kupffer and endothelial cells from rat liver.
Soluble circulating immunoglobulin G immune complexes are mainly eliminated by the liver, predominantly by uptake in the Kupffer cells, but also the liver endothelial cells seem to be of importance. In the present study we have followed the intracellular turnover of immune complexes after Fc(gamma) receptor mediated endocytosis in cultured rat liver endothelial cells and Kupffer cells by means of isopycnic centrifugation, DAB cross-linking and morphological techniques. For the biochemical experiments the antigen, dinitrophenylated bovine serum albumin (BSA), was labeled with radioiodinated tyramine cellobiose that cannot cross biological membranes and therefore traps labeled degradation products at the site of formation. The endocytic pathway followed by immune complexes was compared with that followed by scavenger receptor ligands, such as formaldehyde treated BSA and dinitrophenylated BSA, and the mannose receptor ligand ovalbumin. Both Kupffer cells and liver endothelial cells took up and degraded the immune complexes, but there was a clear delay in the degradation of immune complexes as compared to degradation of ligands taken up via scavenger receptors. The kinetics of the endocytosis of scavenger receptor ligand was unaffected by simultaneous uptake of immune complexes. Experiments using both biochemical and morphological techniques indicated that the delayed degradation was due to a late arrival of the immune complexes at the lysosomes, which partly was explained by retroendocytosis of immune complexes. Electron microscopy studies revealed that the immune complexes were retained in the early endosomes that remained accessible to other endocytic markers such as ovalbumin. In addition, the immune complexes were seen in multivesicular compartments apparently devoid of other endocytic markers. Finally, the immune complexes were degraded in the same lysosomes as the ligands of scavenger receptors. Thus, immune complexes seem to follow an endocytic pathway that is kinetically or maybe morphologically different from that followed by scavenger and mannose receptor ligands.
2,4-Dinitrophenol↗
Response to editorial on air medical crashes
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Journal Article↗
Protein analysis using enzymes immobilized to paramagnetic beads.
A new method combining protein chemistry with enzymes immobilized to paramagnetic beads is presented. The immobilized enzymes can substitute for regular enzymes in a number of protein chemistry protocols, resulting in faster reaction times, less sample contamination, and improved interfacing to modern procedures, like mass spectrometry. Trypsin was used as a model enzyme to test the amount of protein coupled to glass beads and the degree of autodigestion when analyzed by MALDI-MS and HPLC. Immobilization of trypsin resulted in digestions comparable with standard solution digestions using fetuin as a model substrate. Furthermore, fetuin was used to test the stability of the enzyme-coated beads. No apparent loss of enzyme activity was observed after 10 times reuse of trypsin-coated beads. Immobilization of exo- and endoglycosidases to paramagnetic beads resulted in high sensitivity, faster sequential glycosidase digestion of glycopeptides, and reduced sample contamination. All digestions could be performed in less than 24 h, when a tryptic glycopeptide from human lung proteinosis surfactant protein A was used as model compound.
Animals↗
Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes.
Endocytosis may originate both in coated pits and in uncoated regions of the plasma membrane. In hepatocytes it has been shown that fluid phase endocytosis (here defined as 'pinocytosis') is unaffected by treatments that arrest coated pit-mediated endocytosis, indicating that pinocytosis is primarily a clathrin-independent process. In this study we have tried to determine possible connections between pinocytosis and clathrin-dependent endocytosis in rat hepatocytes by means of subcellular fractionation, electron microscopy, and by assessing the influence of inhibitors of clathrin-dependent endocytosis on pinocytosis. As marker for clathrin-dependent endocytosis was used asialoorosomucoid (AOM) labelled with [(125)I]tyramine cellobiose ([(125)I]TC). [(125)I]TC-labelled bovine serum albumin ([(125)I]TC-BSA) was found to be a useful marker for pinocytosis. Its uptake in the cells is not saturable, and any remnants of [(125)I]TC-BSA associated with the cell surface could be removed by incubating the cells with 0.3% pronase at 0 degrees C for 60 min. The data obtained by electron microscopy and by subcellular fractionation suggested that early after initiation of uptake (<15 min) [(125)I]TC-BSA and [(125)I]TC-AOM were present in different endocytic vesicles. The two probes probably join prior to their entrance in the lysosomal compartment. The relation between endocytosis via coated pits and pinocytosis was also studied with techniques that induced a selective density shift either in the clathrin-dependent pathway (by AOM-HRP) or in the pinocytic pathway (by allowing uptake of AuBSA). Both treatments indicated that the two probes ([(125)I]TC-AOM and [(125)I]TC-BSA) were early after uptake, at least partly, in separate endocytic compartments. The different distribution of the fluid phase marker and the ligand (internalised via coated pits) was not due to a difference in the rate at which they enter a later compartment, since a lowering of the incubation temperature to 18 degrees C, which should keep the probes in the early endosomes, did not affect their early density distribution. Incubation of cells in a hypertonic medium reduced uptake both of [(125)I]TC-AOM and [(125)I]TC-BSA; the uptake of [(125)I]TC-AOM was, however, reduced much more than that of the fluid phase marker. This finding supports the notion that the two probes enter the cells via different routes.
Animals↗