Our experiences with 1D-IEF in selection of bone marrow donors.
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Bone Marrow↗
Biomedical subjects
J Suttnar
Publications and source records attributed to J Suttnar.
Separation used for purification of recombinant proteins.
The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.
Chromatography, Liquid↗
Simultaneous isolation of protein C activator, fibrin clot promoting enzyme (fibrozyme) and phospholipase A2 from the venom of the southern copperhead snake.
The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal-chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A2.
Agkistrodon↗
Solid-phase extraction in malondialdehyde analysis.
Malondialdehyde (MDA) has been widely used as an index of lipoperoxidation in biological and medical sciences as well as in the food industry. A solid-phase extraction (SPE) of the condensation product of the MDA with 2-thiobarbituric acid (TBA) was developed using LiChrolut C18ec, 200 mg (Merck, Darmstadt, Germany), as a SPE cartridge and methanol as an eluent for sample pretreatment before HPLC analysis. The samples of blood plasma, platelet concentrates, or erythrocyte membranes (ghosts) were deproteinized by acetonitrile in the presence of sodium hydroxide prior to the reaction with TBA. The reaction mixture was processed using SPE. The SPE extracts (800 microL of methanol) were put to dryness and after dissolution with 100 microliters of mobile phase, 50 microliters was analyzed by RP-HPLC with fluorescence detection (excitation at 514 nm, emission at 556 nm). The mean MDA concentration in plasmas of 32 healthy donors was 0.37 +/- 0.25 mumol/L and the mean MDA concentration in normal ghosts was 8.3 +/- 4.1 pmol/microgram of protein content. In the case of a patient with a severe form of beta-thalassemia, the concentration of plasma MDA was raised to 1.22 mumol/L and the amount of MDA in erythrocytal ghosts was raised to 21.05 pmol/microgram of protein content. MDA concentration in platelet concentrates (six bags) in the first day of storage was 0.46 +/- 0.18 mumol/L and in the fifth day of storage was 0.55 +/- 0.44 mumol/L.
Blood Platelets↗
[Analysis of HLA class I molecules using isoelectric focusing. Its application in selection of bone marrow donors].
BACKGROUND: Differences between HLA proteins class I are assessed by serological typing using HLA allo-antisera. This method suffices for the assessment of HLA signs in allogenic transplantations of bone marrow in genotypically identical siblings. However, it does not suffice in the selection of donors from the wider family of a related or not related donor from the register of voluntary bone marrow donors. In order to assess the polymorphism of the HLA-class I not detected by serological typing other more sensitive techniques are introduced. In the authors department one-dimensional isoelectric focusing was introduced as an auxiliary method for evaluation of identity of molecules in HLA-class I between bone marrow recipient and donor. METHOD AND RESULTS: The authors introduced the method of one-dimensional isoelectric focusing which uses for detection of molecules of HLA-class I a polyclonal antibody against heavy chains of HLA molecules class I and a secondary antibody labelled by alkaline phosphatase. Forty-one pairs of bone marrow donors and recipient were examined. In six instances, i.e. in almost 15%, the authors detected by isoelectric focusing disagreement in HLA molecules class I. The authors present three interesting pairs of donors and recipients where isoelectric focusing helped with the selection of a suitable bone marrow donor. CONCLUSIONS: The isoelectric focusing is at present another method which helps to reveal differences in HLA class I molecules. When methods of DNA analysis are introduced to recognize alleles of the HLA-I class, correlation with serologically and biochemically assessed variants wil be an essential guide in the evaluation of the final typing of HLA molecules class I.
Bone Marrow Transplantation↗
Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation.
Platelet fibrin(ogen) adhesive interactions were investigated in whole citrated blood using the rectangular perfusion chamber at wall shear rates of 300 and 1600 s(-1) with regard to the amount and structure of immobilized protein. Only single platelets adhered to adsorbed fibrinogen at both low and high surface fibrinogen concentrations and at 1600 s(-1) almost no adhesion was observed. When using spray-immobilized protein, platelet adhesion was significantly higher than to adsorbed protein. Conversion of adsorbed fibrinogen to fibrin monomer resulted in the formation of pronounced platelets aggregates and with the elevation of wall shear rate 50% decrease of adhesion took place. Degree of platelet adhesion to fibrin monomer was significantly influenced by immobilized protein concentration at both shear rates. However, the morphology (small and dense platelet aggregates) and extent of platelets adhered to fibrin pentamer was nearly the same at both shear rates. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen fibrin pentamer was prepared using the stepwise synthesis. This methodology is based on the observation that at low concentration immobilized fibrin monomer binds fibrinogen in 1:1 molar ratio. The gradually formed fibrin of a defined size and composition can be a useful tool in the further understanding of the role of fibrin architecture in the pathophysiology of thrombosis.
Adsorption↗
Characterization of platelet antigen for CD45RA monoclonal antibodies.
CD45RA monoclonal antibodies recognize the higher molecular weight isoforms (220 and 205 kDa) of leukocyte common antigen family (CD45), which are typically expressed on B cells and unstimulated T cells. We have found that there are at least three distinct CD45RA monoclonal antibodies which react with platelet 42 kDa (P42) intracellular protein antigen, which seems to be different from any to date described platelet proteins with similar molecular weight. This platelet antigen is a single chain protein, very likely not a glykoprotein, with isoelectrical point between 6.8 and 7.5. P42 does not seem to be a membrane protein and is not associated with platelet cytoskeleton. Results of immunofluorescence assay suggest that P42 may be redistributed to platelet surface after platelet aggregation.
Animals↗
[Diagnosis of beta-thalassemia on the basis of HbA2 determination].
The increased level of HbA2 is a reliable marker of heterozygous beta-thalassaemia. The levels of HbA2 measured by three different methods were compared and the ranges for the normal and for the heterozygous beta-thalassaemia were assessed. The levels of HbA2 2.76 +/- 0.47% for normal (30 blood donors) and 4.62 +/- 0.77% for beta-thalassaemia (50 patients) were obtained by the chromatographic method 2.61 +/- 0.42% HbA2 for normal (30 blood donors) and 5.82 +/- 0.89% HbA2 for beta-thalassaemia (46 patients) were assessed by electrophoresis on hydragel (Sebia) and 2.8 +/- 0.62% HbA2 for normal (30 blood donors) and 6.04 +/- 0.96% HbA2 (47 patients) were found when using cellulose acetate electrophoresis. An increased level of foetal Hb was found in nine patients with beta-thalassaemia. The diagnosis of beta-thalassaemia was confirmed by molecular genetic methods in all cases with an elevated HbA2 level, while a normal HbA2 level did not rule out heterozygous beta-thalassaemia.
Chromatography↗
[Structural variants in hemoglobin occurring in the Czech Republic].
The authors present a review of clinical and laboratory findings of seven in the Czech Republic hitherto diagnosed structural haemoglobin variants. Unstable variants are found most frequently: Hb-Köln, Hb-St. Louis, Hb-Nottingham, Hb-E and Hb-Hradec Králové. The variant Hb-Hradec Králové (Hb-HK) or alpha 2 beta 2 115 (G17) Ala-Asp was newly detected. The great instability of Hb-HK chains makes classical diagnosis of Hb-pathy impossible. It was possible to identify it only at a molecular genetic level. A manifestation of Hb-HK instability is also the thalassaemic feature of the disease and the formation of Heinz bodies from free chains. The only representative of haemoglobins with a high oxygen affinity identified in this country was newly detected. It was given the name Hb-Olomouc or alpha 2 beta 2 86 (F2) Ala-Asp. This haemoglobin variant leads to erythrocytosis in father and son and the same clinical manifestations were recently described also in Japan. The last structural variant of haemoglobin found in this country is Hb-M Milwaukee or alpha 2 beta 2 67 (E11) Val-Glu which in our patients is manifested rather by haemolysis with formation of Heinz bodies than classical cyanosis. The cause of instability of Hb-M in our patients is not known. Hb-S was not diagnosed so far in the Czech Republic.
Adolescent↗
[The fluorescence flow cytometry technique in the diagnosis of hereditary platelet disorders--a case of Glanzmann's thrombasthenia].
The authors describe a method for the diagnosis of heredital platelet membranopathies by means of monoclonal antibodies against the main membrane glycoproteins of thrombocytes, glycoprotein Ib and IIIa. The platelets are differentiated in the flow fluorocytometer from other blood cells by the typical optic profile caused by their size and granular character. Monoclonal antibodies are bound to the appropriate membrane glycoprotein and their amount is then detected by means of a secondary antibody labelled with fluorescein. The intensity of fluorescence of individual platelets is proportional to the number of molecules of the appropriate glycoprotein on their surface. By the above technique a case of Glanzmann's thrombasthenia was diagnosed, a rare hereditary haemorrhagic disease, characterized by the absence or abnormal function of glycoprotein complex IIb/IIIa the platelet receptor for fibrinogen.
Antibodies, Monoclonal↗
EDMA 2000 as a matrix for high-performance liquid chromatography of human haemoglobin chains.
An ethylenedimethacrylate polymer-based matrix (EDMA 2000) is described that shortens the time for separation of haemoglobin (Hb) chains to 30 min, compared with the use of C4 large-pore Vydac columns, commonly used for reversed-phase HPLC of Hb variants. EDMA 2000 was successfully used for the isolation of an abnormal beta-chain of the unstable Hb recently characterized as Hb Nottingham.
Child↗
Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger.
Using Escherichia coli system expressing papilloma virus HPV16 E7MS2 fusion protein as a model system, a novel procedure was applied to solubilize, purify and refold recombinant proteins from E. coli inclusion bodies. The necessity to reactivate proteins at low protein concentrations (owing to their tendency to aggregate at high concentrations) was overcome by solubilization of inclusion bodies in alkaline solution and immobilization of proteins on a strong and resistant anion exchanger. This procedure has an inherent advantage of combining refolding and purification procedures in one step. The solubilization of the fusion protein in an alkaline reagent with the use of an anion exchanger resulted in considerable purification of the recombinant protein at a fairly high concentration. The protein was soluble under mild conditions and reacted with antibodies against the "native" papilloma virus.
Anion Exchange Resins↗
Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F+ expression system inducible by lactose and isopropyl-beta-D-thiogalactopyranoside.
The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a transformable F- strain HB101, is described. These new HB101 (F+) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions. A relatively simple two-step purification of part of a protein (M(r) 27,000) encoded by the gag gene of HIV-1 in this expression system is described. The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate. The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves. Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0. The purified protein did not cross-react with antibodies to E. coli.
Antibodies, Bacterial↗
Hb Nottingham or alpha 2 beta 2 98 (FG5) Val-->Gly in a Czech child.
We report a fourth case of Hb Nottingham [alpha 2 beta 2 98 (FG5) Val-->Gly] observed in an 8-year-old girl in the Czech Republic with clinical and laboratory symptoms of severe hemolytic anemia. The unstable hemoglobin probably represents a de novo mutation, since the parents of the patient and the two siblings do not exhibit any hematological abnormalities. Splenectomy had a beneficial effect on the degree of hemolysis, as well as on the Hb level.
Amino Acid Sequence↗
[Fibrinogen seen as an independent cardiovascular factor from the molecular aspect].
The blood protein fibrinogen is one of the main independent cardiovascular risk factors and it is very likely that an increased fibrinogen level is not the consequence of cardiovascular disease but its direct cause. Fibrinogen participates actively in many processes in the organism and undergoes various changes of the molecule but it is not known what is the molecular background, why even a slight increase of the fibrinogen level is so dangerous as regards increased risk of cardiovascular attacks. In the present work the authors compared, using monoclonal antibodies, the rate of release of fibrinopeptides A and B (FpA, FpB) by the action of thrombin from fibrinogen in solution and from fibrinogen adsorbed to a solid surface. The authors revealed that the rate of FpB breakdown from sorbed fibrinogen, contrary to fibrinogen in solution is comparable to the release rate of FpA and does not depend on the release of FpA (except for competitive inhibition). The release of FpB from sorbed fibrinogen thus takes place, contrary to fibrinogen in solution, at a significant recordable rate from the very onset of thrombin action. Fibrinogen adsorption to a solid surface is associated with conformation changes which cause this effect and which lead to the formation of a two-dimensional formation formed by fibrinogen after its interaction with the surface of activated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
Cardiovascular Diseases↗
[New methods of isolating recombinant proteins].
Recombinant proteins are isolated from very complex protein mixtures present in the producing cell. The isolation process involves in general four mutually interconnected stages: 1. release of the recombinant protein from the cellular environment, 2. preparation of the specimen for separation, 3. separation, 4. qualitative and quantitative analysis of the preparation. Each of these stages is formed by a complex series of methods which destroy the cellular wall, solubilize the specimen and involve the use of suitable precipitation, chromatographic, electrophoretic, immunochemical and other techniques for separation and analysis. In the submitted paper the authors describe a relatively simple isolation of recombinant peptide used for the preparation of a diagnostic kit for AIDS. From knowledge of the sequence of nucleotides in cDNA antisense peptides can be derived which have a high affinity with the isolated protein and they can be also used for affinity chromatography. An effective isolation technique is the use of mimetic ligands on the basis of textile dyes. By their combination and possible modification it is also possible to achieve separation of the required protein from contaminating substances. The mentioned highly specific methods can be combined with classical chromatographic techniques. During every step individual chromatographic fractions are tested by SDS electrophoresis in polyacrylamide gel incl. possible use of immunoblotting a specific staining.
Escherichia coli↗
Affinity high-performance liquid chromatography on heparin-Glc-Spheron.
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Antithrombin III↗
Versatile modular microelectrophoresis system and the simultaneous preparation of large numbers of polyacrylamide gels.
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Animals↗