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Biomedical subjects

A Pol

Publications and source records attributed to A Pol.

At least 37 records · Page 2Linked to original sources
PubMed1999

The "early-sorting" endocytic compartment of rat hepatocytes is involved in the intracellular pathway of caveolin-1 (VIP-21).

The sinusoidal plasma membrane of the hepatocyte is organized into functional and structural microdomains whose origin, maintenance, and functioning are closely related with the endocytic compartment. Three different subcellular fractions, from rat liver, containing caveolin-1, the structural protein of caveolae, were morphologically and biochemically characterized. A caveolae-enriched plasma membrane fraction (CEF), contains large membrane structures surrounding attached internal plasmalemmal vesicles; the receptor-recycling compartment (RRC), contains tubules and vesicles with similar morphology to the internal vesicles observed by electron microscopy in CEF; and finally, caveolin-1 was also detected in early-sorting endosomes (CURL, compartment of uncoupling receptors and ligands). In this study, we show that following an intravenous administration of retinol-binding protein (RBP), there was a redistribution of caveolin-1 from the plasma membrane (CEF) to intracellular endocytic compartments (RRC and early-sorting endosomes). Thus, these results indicate that, in the hepatocyte, caveolae are dynamic structures actively interacting with the endocytic compartment.

Animals↗
PubMed1999

Anaerobic versus aerobic degradation of dimethyl sulfide and methanethiol in anoxic freshwater sediments.

Degradation of dimethyl sulfide and methanethiol in slurries prepared from sediments of minerotrophic peatland ditches were studied under various conditions. Maximal aerobic dimethyl sulfide-degrading capacities (4.95 nmol per ml of sediment slurry. h-1), measured in bottles shaken under an air atmosphere, were 10-fold higher than the maximal anaerobic degrading capacities determined from bottles shaken under N2 or H2 atmosphere (0.37 and 0. 32 nmol per ml of sediment slurry. h-1, respectively). Incubations under experimental conditions which mimic the in situ conditions (i. e., not shaken and with an air headspace), however, revealed that aerobic degradation of dimethyl sulfide and methanethiol in freshwater sediments is low due to oxygen limitation. Inhibition studies with bromoethanesulfonic acid and sodium tungstate demonstrated that the degradation of dimethyl sulfide and methanethiol in these incubations originated mainly from methanogenic activity. Prolonged incubation under a H2 atmosphere resulted in lower dimethyl sulfide degradation rates. Kinetic analysis of the data resulted in apparent Km values (6 to 8 microM) for aerobic dimethyl sulfide degradation which are comparable to those reported for Thiobacillus spp., Hyphomicrobium spp., and other methylotrophs. Apparent Km values determined for anaerobic degradation of dimethyl sulfide (3 to 8 microM) were of the same order of magnitude. The low apparent Km values obtained explain the low dimethyl sulfide and methanethiol concentrations in freshwater sediments that we reported previously. Our observations point to methanogenesis as the major mechanism of dimethyl sulfide and methanethiol consumption in freshwater sediments.

Aerobiosis↗
PubMed1999

Role of methanogens and other bacteria in degradation of dimethyl sulfide and methanethiol in anoxic freshwater sediments.

The roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 micromol of DMS was stoichiometrically converted into 112 micromol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments.

Anaerobiosis↗
PubMed1999

Isolation and characterization of Methanomethylovorans hollandica gen. nov., sp. nov., isolated from freshwater sediment, a methylotrophic methanogen able to grow on dimethyl sulfide and methanethiol.

A newly isolated methanogen, strain DMS1(T), is the first obligately anaerobic archaeon which was directly enriched and isolated from a freshwater sediment in defined minimal medium containing dimethyl sulfide (DMS) as the sole carbon and energy source. The use of a chemostat with a continuous DMS-containing gas stream as a method of enrichment, followed by cultivation in deep agar tubes, resulted in a pure culture. Since the only substrates utilized by strain DMS1(T) are methanol, methylamines, methanethiol (MT), and DMS, this organism is considered an obligately methylotrophic methanogen like most other DMS-degrading methanogens. Strain DMS1(T) differs from all other DMS-degrading methanogens, since it was isolated from a freshwater pond and requires NaCl concentrations (0 to 0.04 M) typical of the NaCl concentrations required by freshwater microorganisms for growth. DMS was degraded effectively only in a chemostat culture in the presence of low hydrogen sulfide and MT concentrations. Addition of MT or sulfide to the chemostat significantly decreased degradation of DMS. Transient accumulation of DMS in MT-amended cultures indicated that transfer of the first methyl group during DMS degradation is a reversible process. On the basis of its low level of homology with the most closely related methanogen, Methanococcoides burtonii (94.5%), its position on the phylogenetic tree, its morphology (which is different from that of members of the genera Methanolobus, Methanococcoides, and Methanohalophilus), and its salt tolerance and optimum (which are characteristic of freshwater bacteria), we propose that strain DMS1(T) is a representative of a novel genus. This isolate was named Methanomethylovorans hollandica. Analysis of DMS-amended sediment slurries with a fluorescence microscope revealed the presence of methanogens which were morphologically identical to M. hollandica, as described in this study. Considering its physiological properties, M. hollandica DMS1(T) is probably responsible for degradation of MT and DMS in freshwater sediments in situ. Due to the reversibility of the DMS conversion, methanogens like strain DMS1(T) can also be involved in the formation of DMS through methylation of MT. This phenomenon, which previously has been shown to occur in sediment slurries of freshwater origin, might affect the steady-state concentrations and, consequently, the total flux of DMS and MT in these systems.

Base Sequence↗
PubMed1999

Acute aortic endocarditis with annular destruction: assessment of surgical treatment with cryopreserved valvular homografts.

BACKGROUND AND AIM OF THE STUDY: Valve ring abscesses in acute infective aortic endocarditis have a low, though not insignificant, prevalence. Surgical treatment with implantation of prosthetic valves may lead to major life-threatening complications, such as recurrent endocarditis and partial or complete prosthetic dislocation. Valvular homografts may offer a higher resistance to recurrent infection and have thus become recognized as an excellent and safe substitute for orthotopic left ventricular outflow reconstruction. METHODS: Between May 1991 and July 1996, 25 patients underwent surgical treatment for aortic endocarditis with annular destruction. Staphylococcus spp. were present in 32% of patients and Streptococcus spp. in 48%. Seven aortic valve replacements (AVR) and 18 aortic root replacements (ARR) were performed using cryopreserved valvular homografts. All grafts were implanted in the subannular position. RESULTS: The overall outcome was good in 23 patients, two died in the early postoperative period. Mean follow up was 38 +/- 18 months (range: 14 to 76 months). No recurrence of endocarditis was detected and no significant alterations of the implants were described. Transvalvular gradients were significantly lower in ARR patients than in AVR patients. CONCLUSIONS: Despite the severity of the tissue damage, cryopreserved homografts, when implanted in the subannular position, constitute a safe and reproducible surgical treatment of aortic endocarditis with annular involvement.

Acute Disease↗
PubMed1999

Regional capnometry with air-automated tonometry detects circulatory failure earlier than conventional hemodynamics after cardiac surgery.

UNLABELLED: Gastrointestinal automated online air tonometry has been proposed for monitoring gastric perfusion in patients at risk of circulatory failure (CF) after cardiopulmonary bypass. In this study, CF was prospectively defined as the requirement for vasoactive support to maintain mean arterial pressure > or = 70 mm Hg after optimization of preload. Hemodynamic variables--oxygen (O2) delivery (DO2), O2 uptake (Vo2), venous-to-arterial [P(v-a)CO2], gastric-to-arterial [P(r-a)CO2], and gastric-to-end-tidal [P(r-et)CO2]Pco2 gap-were retrospectively compared in 14 patients with or without CF during a 12-hr postbypass period (HO-H12). In contrast to patients without CF (n = 7), in patients with CF (n = 7) increased VO2 was not associated with an increase in DO2. P(r-a)CO2 was larger at H0 in CF patients and was the only variable that differed between the two groups. P(v-a)CO2 did not vary significantly in both groups, whereas P(r-a)CO2 increased to a larger extent from H0 to H12 in patients with CF, suggesting selective gastrointestinal hypoperfusion in this group. P(r-et)CO2 provided comparable information to P(r-a)CO2. Hospital length of stay was 4 days longer (P < 0.05) in patients with CF. Increased P(r-a)CO2 and P(r-et)CO2, as monitored with automated air tonometry, were associated with rapid occurrence of CF and prolonged hospital stay after cardiac surgery. IMPLICATIONS: Regional and automated capnometry may be used noninvasively to identify patients at risk of circulatory failure after cardiopulmonary bypass earlier than with conventional variables.

Capnography↗
PubMed1998

Isolated endosomes from quiescent rat liver contain the signal transduction machinery. Differential distribution of activated Raf-1 and Mek in the endocytic compartment.

In this study we identify the molecules involved in the MAPK signal transduction pathway (Ras, Raf-1, Mek, Mek-P and MAPK) in highly purified endosomal fractions isolated from rat liver. Biochemical analysis shows that only the early-sorting endocytic compartment contains activated Raf-1 and Mek. Finally, the exogenous administration of EGF led to redistribution of Raf-1 from the caveolin-enriched plasma membrane into the endosomes.

Animals↗
PubMed1998

Genotypic and phenotypic analysis of the polymorphic thiopurine S-methyltransferase gene (TPMT) in a European population.

1. Characterization of allelic variants of the TPMT gene (TPMT) responsible for changes in TPMT activity, and elucidation of the mechanism by which these alleles act, are required because of the clinical importance of this polymorphism for patients receiving thiopurine drugs. 2. We defined the mutational and allelic spectrum of TPMT in a group of 191 Europeans. Using PCR-SSCP, we screened for mutation the entire coding sequence, the exon-intron boundaries, the promoter region and the 3'-flanking region of the gene. Six mutations were detected throughout the ten exons and seven TPMT alleles were characterized. Four of them, TPMT*2, *3A, *3C and *7, harbouring the known mutations, G238C, G460A, A719G or T681G, were nonfunctional and accounted for 0.5, 5.7, 0.8 and 0.3% of the allele totality, respectively. 3. Within the promoter region, six alleles corresponding to a variable number of tandem repeats (VNTR), were identified. VNTR*V4 and *V5a which harbour four or five repeats of a 17-18 bp unit, were the most frequent (55% and 34%, respectively). The other VNTR alleles, having from five to eight repeats, were rarer. 4. The TPMT phenotype was correctly predicted by genotyping for 87% of individuals. A clear negative correlation between the total number of repeats from both alleles and the TPMT activity level was observed, indicating that VNTRs contribute to interindividual variations of TPMT activity. Therefore, additional analysis of the promoter region of TPMT can improve the phenotype prediction rate by genotyping.

Alleles↗
PubMed1998

Annexin VI defines an apical endocytic compartment in rat liver hepatocytes.

Annexin VI has been demonstrated previously to be a marker for hepatic endosomes. By western blotting with an affinity purified anti-annexin VI antibody it was shown that annexin VI was present in the three morphologically and functionally different endosomal fractions from rat liver. We have quantified the gold-labeled endosomes by immunoelectron microscopy in ultrathin Lowicryl sections of rat liver and now demonstrate that 80% of the total labeling with anti-annexin VI was associated with endocytic structures surrounding the bile canaliculus, the apical domain of hepatocytes, whereas only 20% was found in the subsinusoidal endosomes. In double immuno-gold labeling experiments 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization with antibodies to the polymeric immunoglobulin receptor. Finally, we demonstrate that 50% of endosomes containing internalized gold-labeled transferrin were double labeled with anti-annexin VI antibodies. Thus, annexin VI becomes the first known structural protein at the apical 'early' endocytic compartment of the hepatocyte that may be involved in the receptor recycling and transport to late endocytic/lysosomal compartment pathways.

Animals↗
PubMed1997

Identification of cytoskeleton-associated proteins in isolated rat liver endosomes.

The polypeptides of three highly purified endosomal fractions isolated from the livers of oestradiol-treated rats were analysed by Western blotting, and the amount and distribution of intrinsic and cytoskeletal-associated proteins were quantified and studied. The 'late' endosomes [multivesicular bodies (MVBs)] had the lowest content of cytoskeletal-associated proteins, the most significant being the presence of 25% of the total dynein found in endosomes. The 'early' endosome [compartment of uncoupling receptors and ligands (CURL)] fraction contained kinesin (40% of the total in endosomes), dynein (23%), actin (15%) and tubulin (10%). The receptor-recycling compartment (RRC), also demonstrated to be involved in transcytosis, contained the largest number and enrichment of cytoskeletal proteins: actin (84% of the total in endosomes), alpha-actinin (90%), dynein (52%), tubulin (91%) and kinesin (45%). We also analysed and compared the presence of different endosomal markers such as Rab4, Rab5 and cellubrevin (vesicle soluble NSF attachment protein receptor) in CURL (41%, 15% and 60%) and in RRC (44%, 75% and 30% respectively). Finally, the expression of annexins I, II, IV and VI was studied: annexin I was equally distributed between MVBs and CURL; annexin II was highly enriched in RRC (95%), annexin IV was equally distributed between CURL and RRC, and annexin VI was enriched in CURL (57%). The results indicate that isolated rat liver endosomes contain all the required molecular machinery for the achievement of their role in intracellular trafficking.

Animals↗
PubMed1997

Identification and distribution of proteins in isolated endosomal fractions of rat liver: involvement in endocytosis, recycling and transcytosis.

1. The polypeptides of the three endosomal fractions isolated from livers of oestradiol-treated rats were analysed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approx. 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. 2. The 'early' endosomes [compartment of uncoupling of receptors and ligands (CURL)] comprised the most complex fraction and contained most of the polypeptides found in the 'late' endosomes [multivesicular bodies (MVBs)] and the receptor recycling compartment (RRC). When CURL was analysed by two-dimensional gel electrophoresis after partition with Triton X-114, it showed the largest number of integral membrane polypeptides. 3. Some of the major receptors (polymeric immunoglobulin receptor, transferrin receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, beta1-integrin, mannose 6-phosphate receptor, epidermal growth factor receptor and AGp110) and internalized ligands (IgA, IgG, albumin, haptoglobin, transferrin and alpha2-macroglobulin) were further studied by Western blotting. 4. The distribution of the identified receptors and ligands among the three endosomal fractions was in agreement with their expected functionalities. 5. The polypeptide composition of the bile was also examined and compared with ligands and proteins identified in the different endocytic fractions. 6. Finally, an electron microscopy study confirms the distinctive physical and ultrastructural features of the three isolated endosomal fractions.

Animals↗
PubMed1997

Membrane transport in rat liver endocytic pathways: preparation, biochemical properties and functional roles of hepatic endosomes.

The endocytic compartment has emerged as a major regulator of the uptake and processing of circulating ligands, and has been extensively studied during the last decade. In this work, the polypeptides of the three endosomal fractions: compartment of uncoupling receptors and ligands (CURL), multivesicular bodies (MVB) and receptor recycling compartment (RRC), isolated from livers of estradiol-treated rats, were analyzed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approximately 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. The polypeptide composition of the bile was also studied and compared with ligands and proteins identified in the different endosomal fractions. One- and two-dimensional gel electrophoresis and Western blotting were used to investigate the protein composition of the three isolated endocytic fractions and 39 proteins were identified. The distribution of identified receptors, ligands and structural proteins among the three endosomal fractions was in agreement with their expected functionalities and with the different endocytic pathways in the hepatocyte.

Animals↗
PubMed1997

Formation of dimethyl sulfide and methanethiol in anoxic freshwater sediments.

Concentrations of volatile organic sulfur compounds (VOSC) were measured in water and sediment columns of ditches in a minerotrophic peatland in The Netherlands. VOSC, with methanethiol (4 to 40 nM) as the major compound, appeared to be mainly of sediment origin. Both VOSC and hydrogen sulfide concentrations decreased dramatically towards the water surface. High methanethiol and high dimethyl sulfide concentrations in the sediment and just above the sediment surface coincided with high concentrations of hydrogen sulfide (correlation factors, r = 0.91 and r = 0.81, respectively). Production and degradation of VOSC were studied in 32 sediment slurries collected from various freshwater systems in The Netherlands. Maximal endogenous methanethiol production rates of the sediments tested (up to 1.44 (mu)mol per liter of sediment slurry (middot) day(sup-1)) were determined after inhibition of methanogenic and sulfate-reducing populations in order to stop VOSC degradation. These experiments showed that the production and degradation of VOSC in sediments are well balanced. Statistical analysis revealed multiple relationships of methanethiol production rates with the combination of methane production rates (indicative of total anaerobic mineralization) and hydrogen sulfide concentrations (r = 0.90) or with the combination of methane production rates and the sulfate/iron ratios in the sediment (r = 0.82). These findings and the observed stimulation of methanethiol formation in sediment slurry incubations in which the hydrogen sulfide concentrations were artificially increased provided strong evidence that the anaerobic methylation of hydrogen sulfide is the main mechanism for VOSC formation in most freshwater systems. Methoxylated aromatic compounds are likely a major source of methyl groups for this methylation of hydrogen sulfide, since they are important degradation products of the abundant biopolymer lignin. Increased sulfate concentrations in several freshwater ecosystems caused by the inflow of water from the river Rhine into these systems result in higher hydrogen sulfide concentrations. As a consequence, higher fluxes of VOSC towards the atmosphere are conceivable.

Journal Article↗
PubMed1997

[Immunohistochemical localization of annexin VI in the endocytic compartment of rat liver hepatocytes].

Annexin VI has been isolated from rat liver endosomes and affinity purified antibodies have been produced. By Western blotting, in rat liver subcellular fractions, anti-annexin VI was demonstrated to recognise a 68 kDa band in the three endosomal fractions. In the present study, immunogold labeling of ultrathin Lowicryl sections of rat liver has been used to get insights into the ultrastructural hepatocyte localization. Although at the immunofluorescence level the staining seemed located at the apical, canalicular plasma membrane, domain of the hepatocytes, the electron microscopy revealed that 80% of the labeling, with the anti-annexin VI antibody was specifically localized not at the plasma membrane but in the close subapical endocytic compartment surrounding the bile canalicular plasma membrane of the hepatocyte. Double immunogold labeling with an anti peptide antibody to Rab5 and anti-annexin VI showed that 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization of annexin VI and structures labeled with antibodies to the polymeric immunoglobulin receptor. The results suggest that annexin VI could be involved in regulating the functioning of this apical compartment in the hepatocyte.

Animals↗
PubMed1996

Relationship between CMV and graft rejection after heart transplantation.

This study, which included 153 heart transplant patients, was designed to determine whether the cytomegalovirus (CMV) status of both donor and recipient may influence graft rejection. The follow-up was 1 year and they all received the same triple-drug immunosuppressive regimen with induction (antilymphocyte serum). There was no difference in the total rejection rate, but an increase in repeated rejection rate was shown in transplant recipients with hearts from CMV seropositive donors (P < 0.05). These data strongly suggest the impact of CMV in enhancement but not in induction of rejection. To prevent iterative rejection in the CMV seropositive donor group, antiviral therapy could be proposed during enhancement of antirejection therapy.

Cytomegalovirus Infections↗