Biomedical subjects

R Bucki

Publications and source records attributed to R Bucki.

14 recordsLinked to original sources

PubMed2007

Release of the antimicrobial peptide LL-37 from DNA/F-actin bundles in cystic fibrosis sputum.

Cationic antibacterial peptides (ABPs) are secreted in the airways and function in the first line of defence against infectious agents. They attack multiple molecular targets to cooperatively penetrate and disrupt microbial surfaces and membrane barriers. Antibacterial properties of ABPs, including cathelicidin LL-37, are reduced in cystic fibrosis (CF) airways as a result of direct interaction with DNA and filamentous (F)-actin. Microscopic evaluation of a mixed solution of DNA and F-actin, after the addition of rhodamine-B-labelled LL-37 peptide, revealed the presence of a bundle structure similar to that present in CF sputum. Analysis of CF sputum after centrifugation showed that LL-37 was mostly bound to components of the pellet fraction containing DNA, F-actin and cell remnants. Factors that dissolve DNA/actin bundles and fluidise CF sputum, such as Dornase alfa (recombinant human DNase I), gelsolin, polyaspartate or their combinations, increased the amount of LL-37 peptide detected in the supernatant of CF sputum. The presence of the bacterial endotoxin lipopolysaccharide (LPS) in CF sputum and the ability of LPS to inhibit the antibacterial activity of LL-37 suggests that inactivation of LL-37 function in CF sputum partially results from its interaction with LPS. LL-37-LPS interaction was prevented by an LPS-binding protein (LBP)-derived peptide known for its ability to neutralise LPS, whereas LBPW91A, a mutant peptide that lacks ability to bind LPS, had no effect. A combination of factors that dissolve DNA/filamentous-actin aggregates together with lipopolysaccharide-binding agents may represent a potential treatment for the chronic infections that occur in cystic fibrosis airways.

Actins↗

PubMed2003

Flavonoid inhibition of platelet procoagulant activity and phosphoinositide synthesis.

Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection. Phosphatidylinositol 4,5-bisphosphate (PIP2) was previously reported to play a direct role in phosphatidylserine (PS) exposure, as a Ca2+ target. Thrombin formation and platelet procoagulant activity are dependent on PS exposure. As flavonoids can inhibit phosphoinositide (PPI) kinases, we examined whether changes in PPI metabolism in flavonoid-treated platelets could be involved in their antiplatelet effects. Treatment with the flavonoids quercetin or catechin reduced PS exposure, thrombin formation, PIP2 level and resynthesis after platelet activation with collagen, thrombin or calcium ionophore. Flavonoids also prevented [Ca2+]i increase induced by collagen, but not by the ionophore. The ability of flavonoids to decrease PS exposure induced by ionophore treatment could result from the diminution of PIP2 levels, whereas PS exposure induced by collagen could also be diminished by flavonoids' effects on calcium signaling dependent on PIP2 hydrolysis. These data favor a role for PIP2 in the antiplatelet effects of flavonoids.

Blood Platelets↗

PubMed2001

Involvement of phosphatidylinositol 4,5-bisphosphate in phosphatidylserine exposure in platelets: use of a permeant phosphoinositide-binding peptide.

During platelet activation, phosphatidylserine (PS) exposure on the extracellular face of the plasma membrane is associated with increased procoagulant activity. PS externalization is generally attributed to an increase in intracellular Ca(2+). Various phospholipid transporters, such as specific scramblases or proteins from the family of multidrug resistance proteins, and cofactors such as phosphatidylinositol 4,5-bisphosphate (PIP2) have been proposed to participate in this process. In this study, we used a membrane-permeant polycationic peptide (RhB-QRLFQVKGRR), derived from the PIP2-binding site of gelsolin (GS 160-169) and linked to rhodamine B, to investigate the role of PIP2 in PS externalization in whole platelets. The peptide penetrated rapidly into the platelets, specifically bound to PIP2, and induced PS exposure to a similar extent as thrombin or collagen, but independently of changes in intracellular Ca(2+) or phosphoinositide 3-kinase activity. A pretreatment of platelets with quercetin, an inhibitor of phosphoinositide metabolism, drastically decreased PS exposure induced by agonists or peptide. In large unilamellar vesicles (LUVs), the presence of PIP2 was strictly required for the induction of scrambling of NBD-labeled phospholipids (PC and PS) by the peptide. In inside-out vesicles from erythrocytes (IOVs), the peptide also induced redistribution of PC and PS. Our data suggest that, in intact platelets, PIP2 acts as a target of polycationic effectors, including Ca(2+), to promote PS exposure. The use of a membrane-permeant and fluorescent peptide which binds to PIP2 is a promising tool to investigate the role of PIP2 in various cellular processes.

4-Chloro-7-nitrobenzofurazan↗

PubMed2001

Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly.

Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP(2) binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca(2+)] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5-20 microm peptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of polyphosphoinositides in vivo.

3T3 Cells↗

PubMed2001

[Current views on function and regulation of Ca2+ levels in cell nuclei].

Ca2+ is involved in the regulation of many events in the nucleus, such as gene expression, DNA replication, DNA repair, chromatin fragmentation in apoptosis, modulation of an intranuclear contractile system. In some cases, the function of Ca2+ is mediated by calmodulin. However, the regulation of nucleoplasmic Ca2+ concentration has not been explored thoroughly. The data discussed in this review show that the [Ca2+]n may be regulated independently of that of cytosolic Ca2+. IP3 and cyclic ADP-ribose are the major factors responsible for Ca2+ release into the nucleus from perinuclear space.

Animals↗

PubMed2000

Phosphatidylinositol 4,5-bisphosphate domain inducers promote phospholipid transverse redistribution in biological membranes.

Transmembrane phospholipid redistribution (scrambling), leading to exposure of phosphatidylserine on the cell surface, plays a physiological role to induce platelet procoagulant activity and clearance of injured or apoptotic cells. Scrambling is generally attributed to an increase in intracellular Ca(2+) and would be mediated by a protein (scramblase), whose activity could be modulated by cofactors. We reported previously that phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a positive regulator of Ca(2+)-induced scrambling. We show here, using inside-out vesicles from erythrocyte membranes, that a pleckstrin homology (PH) domain, which interacts with high affinity with PIP(2), inhibited Ca(2+)-induced scrambling, confirming the role of PIP(2). As Ca(2+) is known to interact with PIP(2) and to promote the formation of lateral domains of acidic phospholipids in membranes, we investigated whether PIP(2) domain formation could be involved in scrambling. Spermine, polylysine, and MARCKS (151-175) peptide caused scrambling in parallel to their reported ability to form domains of acidic phospholipids, including PIP(2). Similarly, neomycine, another PIP(2)-interacting polycation, induced scrambling. A PIP(2) antibody was also found to induce scrambling, presumably by a similar mechanism, since phospholipid antibodies are known to promote phospholipid capping. In conclusion, Ca(2+) is not the sole inducer of scrambling, and formation of PIP(2) domains could play a critical role in this process.

Antibodies, Monoclonal↗

PubMed2000

Diabetes affects phospholipid content in the nuclei of the rat liver.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on the content of different phospholipids and the incorporation of blood-borne 14C-palmitic acid into the phospholipid moieties in the rat liver nuclei. Diabetes was produced by intravenous administration of streptozotocin, and determinations were carried out two and seven days thereafter. Phospholipids were extracted from isolated nuclei and separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. Following that, they were quantified and radioactivity was measured. It was found that, in comparison to non-diabetic controls, two-day diabetes reduced the total content of phospholipids in the nuclei by 9.6%. The content of phospholipids in the nuclei by 9.6%. The content of phosphatidylcholine and phosphatidylserine was reduced and the content of the remaining phospholipids was stable. The specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin, based on radioactivity incorporated from 14C-palmitic acid, was elevated. Seven-day diabetes resulted in a reduction of the total phospholipid content in the nuclei by 39.4%. This was accounted for by a reduction in the content of each phospholipid fraction with the exception of cardiolipin. The specific activity of each phospholipid fraction, was elevated in this group. It is concluded that insulin is involved in the regulation of the nuclear phospholipid content.

Animals↗

PubMed2000

Effect of triiodothyronine on the content of phospholipids in the rat liver nuclei.

The aim of the present study was to examine the effect of triiodothyronine (T3) on the content of phospholipids and on the incorporation of blood-borne palmitic acid into the phospholipid moieties in the nuclei of the rat liver. T3 was administered daily for 7 days, 10 microg x 100 g(-1). The control rats were treated with saline. Each rat received 14C-palmitic acid, intravenously suspended in serum. 30 min after administration of the label, samples of the liver were taken. The nuclei were isolated in sucrose gradient. Phospholipids were extracted from the nuclei fraction and from the liver homogenate. They were separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. The content and radioactivity of each fraction was measured. It was found that treatment with T3 reduced the content of phosphatidylinositol and increased the content of cardiolipin in the nuclear fraction. In the liver homogenate, the content of phosphatidylinositol decreased and the content of phosphatidylethanolamine and cardiolipin increased after treatment with T3. The total content of phospholipids after treatment with T3 remained unchanged, both in the nuclear fraction and in the liver homogenate. T3 reduced the specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin and had no effect on the specific activity of sphingomyelin and phosphatidylinositol both in the fraction of the nuclei and the liver homogenate. It is concluded that excess of triiodothyronine affects the content of phospholipids in the nuclei. The changes in the content of phospholipids in the nuclei largely reflect changes in their content in the liver.

Animals↗

PubMed2000

[The effect of cigarette smoking and alcohol consumption on efficacy of Helicobacter pylori eradication].

None of the drug regimens used to treat H. pylori infection ensures 100% of efficacy. One may think therefore that among factors modifying H. pylori eradication there are also cigarette smoking and alcohol consumption. The aim of the study was to determine the influence of cigarette smoking and alcohol consumption on efficacy of H. pylori eradication. The study was conducted on 142 H. pylori positive peptic ulcer patients treated with either OAT-omeprazole (2 x 20 mg), amoxycillin (2 x 1000 mg), tinidazole (2 x 500 mg) (69 patients) or OAC-omeprazole (2 x 20 mg), amoxycillin (2 x 1000 mg), clarithromycin (2 x 250 mg) (73 patients). Detailed information on smoking and drinking habits was obtained from a questionnaire fulfilled by all subjects. Patients were defined as smokers if smoked 5 or more cigarettes per day and as drinkers if drank 25 g or more alcohol per week. To enter the study patients had to have confirmed H. pylori infection by two tests (CLO-test and histology). Eradication was considered successful if both tests gave negative results 4-6 weeks after the cessation of treatment. The efficacy of H. pylori eradication was similar in both groups (OAT--69.6%, OAC--78.1%). Patients who smoked cigarettes had lower rate of H. pylori eradication in OAC group (smokers 65.8%, non-smokers 91.4%, p < 0.01), while patients who drank alcohol had higher eradication rate in OAT group (drinkers 85.2%, non-drinkers 59.5%, p < 0.05). When two factors (smoking, drinking) were analyzed together, it was found that in drinkers treated with OAT, smoking did not change the efficacy of H. pylori eradication, while in non-drinkers decreased by two times (75.0% vs 38.9%, p < 0.02). In drinkers treated with OAC, smoking did not change the efficacy of H. pylori eradication (but this was likely related to the limited number of patients), while decreased it in non-drinkers from 90.0% to 65.2% (p < 0.05). When two groups were analyzed together (OAT + OAC), the lowest efficacy of H. pylori eradication exhibited smokers who do not drink (53.7%) followed by smokers who drink (75.8%), non-smokers who do not drink (83.3%) and non-smokers who drink (92.9%); in each case the efficacy of eradication was higher than in smokers who do not drink (p < 0.05, p < 0.01, p < 0.01, respectively). Both cigarette smoking and alcohol consumption can affect the efficacy of H. pylori eradication. Smoking and drinking habits should be taken into account, when the set of drugs for H. pylori eradication is chosen.

Adult↗

PubMed1998

Calcium induces phospholipid redistribution and microvesicle release in human erythrocyte membranes by independent pathways.

The increase in intracellular Ca2+ concentration in erythrocytes and platelets results in simultaneous phospholipid scrambling and microvesicle shedding. Microvesicle formation involves membrane fusion events which were proposed either to be tightly linked to phospholipid transversal redistribution or to occur by a separate mechanism. We report here that in erythrocytes incubated in high K+ medium, or in resealed ghosts, phospholipid scrambling can be fully induced by intracellular Ca2+ without microvesicle formation. Furthermore, in ghosts resealed in the presence of spermine, intracellular Ca2+, at low concentration, was able to induce microvesicles, whereas scrambling was drastically inhibited. Surprisingly, in spermine-containing ghosts prepared from erythrocytes of a patient with a bleeding disorder, due to a lack of Ca2+-induced phospholipid scrambling and vesicle shedding (characterized as a Scott syndrome), Ca2+ also promoted microvesicle release. Data show that phospholipid scrambling and microvesicle production, although closely regulated, proceed by independent pathways.

Aged↗

PubMed1997

Effect of increased uptake of plasma fatty acids by the liver on lipid metabolism in the hepatocellular nuclei.

The nucleus contains different lipids. The aim of the present study was to examine whether increased uptake of free fatty acids by the liver affects lipid metabolism in the hepatocellular nuclei. The experiments were carried out on three groups of Wistar rats: I - male, control; II - male, heparin-treated, and III - female. [14C]-palmitic acid suspended in rat donor serum was administered intravenously 5 and 30 min before tissue samples were taken. Lipids were extracted from isolated liver nuclei and separated into different fractions (phospholipids - PH, monoacylglycerols - MG, diacylglycerols - DG, cholesterol - CH, free fatty acids - FFA, triacylglycerols - TG and cholesterol esters - CHE). It was found that 5 min after administration of the label all isolated nuclear lipid fractions were radioactive. Most of the radioactivity was located in the fraction of PH, TG and FFA. Elevation in the plasma FFA concentration (heparin-treated group) resulted in increased incorporation of [14C]-palmitic acid into the nuclear lipids and changes in its distribution. In the female rats the radioactivity of nuclear lipids was higher than in the male-controls. There were also differences in the percentage distribution of the radioactivity in different lipid fractions between the two groups. The concentration of PH and TG in the nuclei increased only in the heparin-treated but not in the female rats. However, specific activity of the nuclear PH and TG increased in with both groups compared to the male-control group. It is concluded that (a) the blood-borne FFA rapidly enter the nuclear lipid pool and (b) increased uptake of the plasma-borne FFA by the liver affects the nuclear lipid metabolism.

Animals↗

PubMed1997

[Lipids in the cell nucleus].

The paper contains available data on the content, composition and metabolism of different lipid fractions in the nuclei. The results on physiological function of the nuclear lipids are also included. Nuclear phosphatidylinositols have been shown to play a role of messengers signalling from the cytoplasm to the nuclei. Most sphingomyelin is located in the nucleoplasm and it effects stability of DNA. A role of nuclear triacylglycerols remains unknown. Polyunsaturated long chain fatty acids control transcription of genes encoding lipogenic and glycolytic enzymes. Cholesterol present in the nuclear envelope plays only structural function, but that present in chromatin may be involved in regulation of cholesterol biosynthesis.

Animals↗

PubMed1997

[Various functions of human erythrocyte membrane lipids].

The major phospholipid classes of the human red blood cell membrane are: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin. These phospholipids are distributed asymmetrically across the two halves of the lipid bilayer. This asymmetry appears to be generated and maintained by an ATP-dependent translocation of aminophospholipids from outer to inner leaflet, and by the interaction of phospholipids with skeletal proteins. The phosphoinositides account for 3-4% of total erythrocyte membrane phospholipid. They play an important role in signal transduction and are involved in other various membrane functions.

Adenosine Triphosphate↗

PubMed1996

The plasma borne free fatty acids rapidly enter the hepatocellular nuclei.

Long-chain free fatty acids (FA) were shown to exert a regulatory function in the nucleus. However, the route of their entry remains uncertain. The aim of the present study was to examine whether the extracellular FA enter the hepatocellular nuclei. The experiments were carried out in vivo and in vitro. Intravenous administration of albumin-bound [14C]-palmitic and [14C]-linoleic acid resulted in rapid accumulation of the labels in the nuclear lipids. Unesterified [14C]-palmitic acid represented 22.4 +/- 1.7 and [14C]-linoleic acid 17.6 +/- 1.3 percent of the total lipid radioactivity. In vitro, confocal laser scanning microscopy was used to examine 12-NBD-stearate (a fluorescent derivative of stearate) translocation into the nuclei of isolated hepatocytes. It was found that 12-NBD stearate enters the nucleus and that this uptake depends on the extracellular and/or cytoplasmic concentration. It is concluded that factors (e.g. dietary) leading to alterations in the plasma FA composition and content can result in rapid changes of the nuclear FA pool and thus regulate certain nuclear processes.

4-Chloro-7-nitrobenzofurazan↗