Double guide wire placement for endoscopic pancreaticobiliary procedures.
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Biliary Tract Diseases↗
Biomedical subjects
M Varsányi
Publications and source records attributed to M Varsányi.
Conservative vs. endoscopic closure of colocutaneous fistulas after percutaneous endoscopic gastrostomy complications.
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Colonic Diseases↗
Calsequestrin: more than 'only' a luminal Ca2+ buffer inside the sarcoplasmic reticulum.
In striated muscle, the sarcoplasmic reticulum (SR) Ca2+ release/ryanodine receptor (RyR) channel provides the pathway through which stored Ca2+ is released into the myoplasm during excitation-contraction coupling. Various luminal Ca2+-binding proteins are responsible for maintaining the free [Ca2+] at 10(-3)-10(-4) M in the SR lumen; in skeletal-muscle SR, it is mainly calsequestrin. Here we show that, depending on its phosphorylation state, calsequestrin selectively controls the RyR channel activity at 1 mM free luminal [Ca2+]. Calsequestrin exclusively in the dephosphorylated state enhanced the open probability by approx. 5-fold with a Hill coefficient (h) of 3.3, and increased the mean open time by about 2-fold, i.e. solely dephosphorylated calsequestrin regulates Ca2+ release from the SR. Because calsequestrin has been found to occur mainly in the phosphorylated state in the skeletal-muscle SR for the regulation of RyR channel activity, the dephosphorylation of calsequestrin would appear to be a quintessential physiological event.
Animals↗
Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing.
Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.
1-Phosphatidylinositol 4-Kinase↗
Does phosphorylase kinase control glycogen biosynthesis in skeletal muscle?
Immunoblotting as well as enzyme assays demonstrate the presence of the self-glucosylating protein, glycogenin, in the protein-glycogen complex, in the sarcoplasmic reticulum and in phosphorylase kinase. In all three compartments glycogenin occurs in different, albeit, defined glucosylated forms, which upon deglucosylation are converted into a 42 kDa form. We suggest that phosphorylase kinase might have a dual function in glycogen biogenesis: firstly, control of glycogen degradation in the protein-glycogen complex via phosphorylation of glycogen phosphorylase b; secondly, regulation of glycogen biosynthesis on the sarcoplasmic reticular membranes via phosphorylation and thereby inhibition of glycogen synthase.
Animals↗
Sarcoplasmic reticular Ca2+ release channel is phosphorylated at serine 2843 in intact rabbit skeletal muscle.
The Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (SR) can be phosphorylated on serine 2843 in isolated heavy SR (HSR) fraction by associated protein kinase(s). Laser desorption mass spectrometry of the major [32P]phosphopeptide demonstrates that in HSR the channel is already phosphorylated before the in vitro phosphorylation starts. However, occasionally ca. 60% of the receptor cannot be phosphorylated, the degree of in vitro phosphorylation is generally low. We suggest an interaction of the channel with a not yet identified effector molecule which might determine the phosphorylation degree in addition to the enzymatic activities of associated kinase(s) and phosphatase(s).
Amino Acid Sequence↗
Enhancement of Ca2+ release channel activity by phosphorylation of the skeletal muscle ryanodine receptor.
The Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (SR) can be phosphorylated by membrane associated protein kinase(s) utilizing endogenously synthesized or exogenously added ATP. The channel protein has been enriched in non-phosphorylated and phosphorylated form from heavy SR following solubilization with CHAPS (3-[(3-cholamidopropyl)dimethylammonio-1-propane-sulfonate) and ultracentrifugation on a linear sucrose/CHAPS gradient. Reconstitution of the isolated channels into planar bilayers shows that phosphorylation enhances the open probability by increasing the sensitivity towards micromolar Ca2+ and ATP. The phosphorylation induced enhancement of the channel activity can be reversed by purified protein phosphatase 2A.
Adenosine Triphosphate↗
Identification of a new 80 k isoform of phosphatidylinositol 4-phosphate 5-kinase from bovine brain.
Phosphatidylinositol 4-phosphate 5-kinase is associated with bovine brain microsomes to an extent of approximately 65% of the total cellular enzyme activity. This membrane-associated kinase activity can be solubilized with Triton X-114. Following polyacrylamide gel electrophoresis in the presence of SDS the enzyme can be renaturated from gel slices in the presence of desoxycholate and Triton X-100. Catalytic activity appears at an apparent molecular weight of 80 k. Analysis of the reaction product formed by the 80 k protein reveals the existence of a 5-phosphotransferase, identifying the 80 k polypeptide as a new phosphatidylinositol 4-phosphate 5-kinase isoform.
Animals↗
Compartmentalized ATP synthesis in skeletal muscle triads.
Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads.
Adenosine Triphosphate↗
The effect of azathioprine, cyclosporine A and insulin on the in vitro lymphocyte-mediated cytotoxicity in type I diabetic patients.
Previously both specific and nonspecific immune reactions have been reported in patients with type I diabetes mellitus. In this study the effect of various immunosuppressive drugs and insulin was studied on in vitro lymphocyte-mediated cytotoxicity in 20 type I diabetic patients. Twenty sex- and age-matched healthy subjects served as controls. Human pancreas-extract (300 micrograms/ml protein)-coated, 51-Chromium labeled chicken erythrocytes were used as target cells and separated T-lymphocytes as effector cells with and without azathioprine 50 micrograms/50 microliters (Wellcome), Cyclosporine A 5 ng/50 microliters (Sandoz) and MC Actrapid insulin 0.1 IU/50 microliters (Novo). The degree of cytotoxicity was expressed with cytotoxic capacity: the number of maximal killed target cells. Simultaneously islet cell antibodies (ICA) in sera and the number of activated T-lymphocytes were assessed. Significant lymphocyte-mediated cytotoxicity was observed in the majority of type I diabetic patients (18/20), while no cytotoxicity was found in the control cases. The cytotoxicity decreased in all 16 patients using azathioprine or insulin, independently of ICA and HLA-DR positivity. The number of killed target cells was lowered considerably by Cyclosporine A in all 18 patients having cytotoxicity against pancreas-extract. Our observations reveal that Cyclosporine A proved to be the most effective immunosuppressive agent in vitro. It decreases not only the leucocyte migration inhibition as previously observed, but also the lymphocyte-mediated cytotoxicity, which represents the late stage of cellular immune reactions against pancreatic tissue.
Adolescent↗
The effect of some drugs on in vitro cellular immune reactions and on circulating immune complexes in patients with myocardial infarction.
The immunomodulatory effect of the lipid-reducing etofibrate, the calcium antagonist nifedipine and the immunosuppressive cyclosporine A was studied on in vitro cellular immune response in the presence of human aortic extract and of human LDL in 56 patients with acute myocardial infarction. 50 sex- and age-matched healthy persons were used as controls. Leukocyte migration test, level of C1q and of immune complexes and serum lipid parameters were measured. Leukocyte migration inhibition was observed against aorta in 68%, against LDL in only 23% of patients, which were significantly reduced by etofibrate and nifedipine and a similar, but not significant tendency was found using cyclosporine A against aorta. Leukocyte migration stimulation was observed in 9.0% and in 27% of patients against aorta and LDL, respectively. They were also considerably decreased by etofibrate and nifedipine. A high level of circulating immune complexes was found in hyperlipemic younger patients together with lower migration inhibition against LDL, which may be explained by authors' hypothesis: LDL is a component of circulating immune complexes, so there are few free LDL for lymphokines in the circulation. A correlation between specific (anti-aorta, anti-LDL) and nonspecific (PHA) immune responses was also detected in patients. The use of these drugs may be useful in the therapy of patients with myocardial infarction in order to modulate cellular immune reactions against vascular wall and LDL.
Aged↗
[Electrochemical or mechanical preparation of metal surfaces of fixed dentures or splints].
The authors describe electrochemical etching and sand-blasting of Ni-Cr alloys (Wiron-88, Nidemet-K) for adhesive bridges.
Dental Abutments↗
Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit.
Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.
Adenosine Triphosphate↗
Intracellular localization of inositol-phospholipid-metabolizing enzymes in rabbit fast-twitch skeletal muscle. Can D-myo-inositol 1,4,5-trisphosphate play a role in excitation-contraction coupling?
Rabbit fast-twitch skeletal muscle microsomes have been separated by isopycnic centrifugation on a linear sucrose gradient into triads and light sarcoplasmic reticulum. In both fractions phosphatidylinositol-kinase activity is found [Varsányi et al. (1986) Biochem. Biophys. Res. Commun. 138, 1395]. In contrast, phosphatidylinositol-4-phosphate kinase is nearly exclusively associated with triads. The phosphatidylinositol-4,5-bisphosphate-phosphodiesterase activity shows a biphasic distribution: approximately 50% of the activity is associated with triads and 50% appears in the overlay. Triads have been broken mechanically into transverse tubules and terminal cisternae, then separated by isopycnic sucrose-gradient centrifugation. Both fractions exhibit phosphatidylinositol-kinase activity; the activities of phosphatidylinositol-4-phosphate kinase and phosphatidylinositol-4,5-bisphosphate phosphodiesterase are associated mainly with the transverse tubules. Consequently, in rabbit fast-twitch skeletal muscle all necessary enzymes for production of D-myo-inositol 1,4,5-trisphosphate are associated with transverse tubules. Phosphatidylinositol-4,5-bisphosphate phosphodiesterase associated with triads shows a pH optimum at 6.8. The enzyme is maximally active between pCa 5 and pCa 4. Mg2+ inhibits the enzyme activity half-maximally at about 1 mM. Guanine-nucleotide-binding proteins seem not to be involved in the regulation of enzyme activity; guanosine 5'-[gamma-thio]triphosphate does not influence phosphatidylinositol-4,5-bisphosphate phosphodiesterase activity. It correlates well with the observation that neither alpha 1-adrenergic nor muscarinic receptors have been found in fast-twitch rabbit skeletal muscle. On basis of the respective enzyme activities estimations on maximal phosphatidylinositol turnover were made and a possible involvement of this signal pathway in excitation-contraction coupling has been discussed. Furthermore, calculations show that during a single twitch D-myo-inositol 1,4,5-trisphosphate concentration does not reach more than 2 nM. However, during a 4-s tetanus D-myo-inositol 1,4,5-trisphosphate can accumulate to a level which could effect force generation [Thieleczek and Heilmeyer (1986) Biochem. Biophys. Res. Commun. 135, 662] and aldolase distribution (Thieleczek et al., unpublished results).
Animals↗
Human pancreatic extract and rat pancreatic islet homogenate-coated chicken erythrocytes as targets of lymphocyte-mediated cytotoxicity in type I diabetic patients.
In this study 51Cr-labeled chicken erythrocytes coated with human pancreatic extract or rat pancreatic islet homogenate served as target cells for the detection of lymphocyte-mediated cytotoxicity in type I-diabetics. T-lymphocytes from type I-diabetic patients showed a significant cytotoxic capacity against target cells coated with human antigens as well as rat pancreatic islet homogenate. The frequency and intensity of cytotoxicity proved to be similar against the two types of targets. Since rat pancreatic islets are easily available and contain more characteristic beta-cell antigens than human pancreas extract, the homogenate of rat islets of Langerhans is more suitable than human pancreatic extract for studying the lymphocyte-mediated cytotoxicity in type I-diabetic patients.
Adolescent↗
Ca2+ regulation of 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate formation and hydrolysis on sarcoplasmic-reticular Ca2+-transport ATPase. A new principle of phospholipid turnover regulation.
Lipid phosphorylation was shown to occur on the isolated sarcoplasmic-reticulum (SR) Ca2+-transport ATPase. More than 95% of the radioactivity incorporated on incubation of the SR ATPase with [gamma-32P]ATPMg can be extracted with acidic organic solvents and was identified as 1-(3-sn-phosphatidyl)-1D-myo-inositol 4-phosphate (PtdIns4P) [Varsányi, Toelle, Heilmeyer, Dawson & Irvine (1983) EMBO J. 2, 1543-1548]. This lipid phosphorylation is only observed at nanomolar concentrations of free Ca2+; in the presence of micromolar free Ca2+ PtdIns4P disintegrates rapidly. Also, upon blockade of the kinase reaction PtdIns4P decomposes, indicating a PtdIns/PtdIns4P turnover. The PtdIns4P concentration is dependent on the free Ca2+ concentration, being half-maximal at 35 nM-Ca2+. PtdIns4P hydrolysis is catalysed by a PtdIns4P phosphomonoesterase; accordingly no diacylglycerol is formed, which would be a product of a phosphodiesteratic cleavage. Fluoride inhibits this phosphomonoesterase. Ca2+ does not influence directly either the PtdIns kinase or the PtdIns4P phosphomonoesterase. PtdIns4P forms a tight complex with the transport ATPase, from which it can be removed only by chromatography on heparin-agarose in the presence of Triton X-100. It is concluded that Ca2+ regulates the PtdIns/PtdIns4P turnover by availability of substrate, depending on the Ca2+-transport-ATPase conformation, which traps or exposes the respective lipid head groups.
1-Phosphatidylinositol 4-Kinase↗
Phosphorylation of phosphatidylinositol associated with the nicotinic acetylcholine receptor of Torpedo californica.
When isolated, detergent solubilized and affinity chromatographically purified nicotinic acetylcholine receptor of Torpedo californica electric organ is incubated with [gamma-32P]ATP/Mg2+, phosphatidylinositol 4-phosphate (PIP) is formed from receptor associated phosphatidylinositol (PI). This receptor associated endogenous kinase activity is enhanced by orthovanadate and, remarkably, also by acetylcholine. Exogenously added PI-kinase only increases the phosphorylation rate if vanadate is present. PIP as the main phosphorylation product (up to 95%) remains bound to the beta-, gamma- and delta-subunits of the receptor and to the receptor associated v-protein. The alpha-subunits do not carry 32p phosphate; no phosphatidylinositol 4,5-bisphosphate formation has been observed. Concomitant to lipid phosphorylation tyrosine and serine residues are phosphorylated (5% of total incorporated 32P phosphate).
1-Phosphatidylinositol 4-Kinase↗
Immune reactions in patients with type I and with type II diabetes mellitus.
Since several data refer to the role of immune processes in the pathogenesis of diabetes mellitus, this study was performed to compare aspecific and specific immune reactions in type I- and in type II diabetic patients over a six month period. The percentage and the absolute number of SRBC-rosette forming active E(A), of theophylline-resistant E(Thr) and of ORCB-rosette forming T(M)-cell subsets proved to be elevated in newly diagnosed type I but reduced in type II diabetic patients. Also an elevated percentage of HLA-DR positive, activated T cells was found in the majority of recent-onset type I diabetics. In the presence of human pancreas extract, a significant inhibition of leucocyte migration, a pronounced and specific cytotoxic capacity of all lymphocyte subsets (especially of the T(G)-cells), and elevated antibody titers (passive haemagglutination, indirect immunofluorescence) were observed in almost all type I diabetics, but only in a few cases of type II patients. After six months, the frequency both of the aspecific and of the specific immune parameters was decreased in type I diabetics, but no changes were observed in the type II diabetics with a previously positive test. The latter patients required insulin therapy at the time of the second investigation. The leucocyte migration inhibition test and the lymphocyte-mediated cytotoxicity are suitable for studying in-vitro-sensitization against pancreatic tissue and they might predict later insulin-dependency in type II diabetic patients.
Adolescent↗