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  • 1980-1984  (1,711)
  • Biochemistry and Biotechnology  (1,710)
  • Nuclear reactions
  • 101
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984), S. 225-236 
    ISSN: 0263-6484
    Keywords: Nucleic acids ; Purkinje cells ; DNA ; cytophotometry ; flow cytometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Several cytochemical studies of the DNA content and ploidy status of neuronal cell nuclei in the central nervous system have reported the occurrence of hyperdiploid amounts of DNA in Purkinje cells and suggest the existence of some type of ‘extra’ DNA, the biological significance of which is, as yet, unknown. To explore this phenomenon further, the DNA content of glial and Purkinje cell nuclei was determined in several vertebrate species, using the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole (DAPI) to stain isolated cerebellar nuclei for analysis with a single parameter flow cytometer. The Feulgen reaction for DNA was used to stain liver and cerebellar tissue imprints for the measurement of individual nuclei with a Vickers M86 integrating microdensitometer. In both types of analyses, chicken erythrocyte nuclei served as an internal reference standard of 2.5 pg DNA per cell. The mean DNA content of Purkinje cells and glial or granule cells was essentially the same as that found for diploid (2C) non-neuronal cells, such as hepatocytes, in rainbow trout, Amazon molly fish, salamander (Plethodon), mouse, rat, rabbit, cat, dog, monkey and human. Although Purkinje cell nuclei with 4C DNA levels were found in all of these species, except salamander and rabbit, the frequency of such cells was low (1-7%) and varied with the species. There was a low incidence of Purkinje cell nuclei with interclass DNA amounts in all species examined. Our data show that most neuronal cell nuclei in the cerebellum contain 2C levels of DNA.
    Additional Material: 9 Ill.
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  • 102
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984), S. 254-256 
    ISSN: 0263-6484
    Keywords: Bones ; 1,3-bisphosphoglycerate ; bisphosphoglycerate mutase ; erythropoiesis ; phosphoglycerate kinase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Rat bone marrow cells have been fractionated by density gradient in Percoll. Differential counting of erythroid cells, haemoglobin concentration and bisphosphoglycerate mutase and phosphoglycerate kinase activities have been determined in cellular fractions. As shown by means of a statistical approach, an increase in bisphosphoglycerate mutase activity and a slight decrease in phosphoglycerate kinase activity is found in erythroid cells as their haemoglobin content increases. Our results suggest that there is a synthesis of 2,3-bisphosphoglycerate during the erythropoietic process which parallels the synthesis of haemoglobin.
    Additional Material: 3 Ill.
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  • 103
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 104
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 1-17 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Additional Material: 12 Ill.
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  • 105
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 26-29 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A widely applicable procedure for electrophoretic separation and quantification of proteins is described. Different colored markers of myoglobin were prepared by carbamylation and those with suitable isoelectric points (pI's) were isolated. Such markers were added to samples of serum and plasma which were examined by improved isoelectric focusing in agarose gel. This facilitated isolation of a certain transferrin with pI 5.7. This protein was quantified in gel pieces by zone immunoelectrophoresis assay. The overall S.D. was 〈10%. Immunofixations were performed to check the quality of focusing and the accuracy of gel punching.
    Additional Material: 2 Ill.
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  • 106
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Rheophoresis, i.e. thin-layer electrophoresis in particulate gels with controlled evaporation, combines in one experiment the chromatographic and the electrophoretic characteristics of macromlecules in such a way that they can be determined independently of each other. In this paper theory and experimental corroboration for molecular size determination are presented. We describe the migration velocity of any molecule as the sum of an electrophoretic, an electroosmotic and a rheophoretic component. The latter is the velocity originating from the liquid flow produced by evaporation. It is a function of the position along the gel axis and the cross-section available to the molecule, but totally independent of the molecular charge. As a result of the position dependence, the rheophoretic component can be isolated from the two others and the available cross-section determined by means of a simple plot. Thus, the chromatographic partition coefficient is readily calculated and the effective hydrodynamic radius of the molecule obtained.
    Additional Material: 3 Ill.
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  • 107
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Group specific component (Gc) is known to interact with both vitamin D3 metabolites and actin. The present study was undertaken to determine if analytical isoelectric focusing could form the basis of a simple and reliable method for discriminating native Gc and complexes with 25-hydroxycholecalciferol (25(OH)D3) from those formed with actin and with both ligands. Increasing amounts of G-actin and/or 25(OH)D3 were added to purified Gc of both Gc1 and Gc2 phenotypes. Actin and 25(OH)D3 interacted independently and simultaneously with native Gc, giving rise to three different complexes of increasing acidity: Gc-25(OH)D3, Gc-actin, and actin-Gc-25(OH)D3, which were clearly resolved from the native Gc protein and from each other. In addition, the inherent differences in microheterogeneity and polymorphism between the two major phenotypes examined (Gc1 and Gc2) were also retained in their respective complexes. Similar results were obtained upon addition of 25(OH)D3 or actin to whole human serum, the bands corresponding to native or complexed Gc being recognized in this case by print immunofixation. These results indicate that complexes formed between Gc protein and both 25(OH)D3 and actin can be clearly detected and resolved by electrophoretic procedures.
    Additional Material: 4 Ill.
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  • 108
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple technique has been developed for the electrophoretic extraction of milligram to microgram quantities of protein, under constant monitoring by dye line, in a concentrated form from polyacrylamide gels run under alkaline, neutral and acidic conditions. Typically, the extraction time from 5 gel slices per tube containing about 1.2 mg of bovine serum albumin or α-chymotrypsinogen A was 3 h, and the maximum concentration of the recovered protein was 5 mg/ml with a total recovery of more than 90% in all cases. Higher protein concentrations (11 mg/ml) could be obtained by increasing the extraction time to 3.5 h or by using resins like glass beads. The alkaline extraction system has been applied to the purification of glutamyl-tRNA synthetase up to homogeneity, and also to ferritin. This technique can also be used under reducing conditions and the scale of operation can be adjusted by the number of tubes or number of slices.
    Additional Material: 9 Ill.
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  • 109
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 35-42 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Proteins visualised by Coomassie Blue staining of two-dimensional polyacrylamide gels of complex mixtures of brain proteins were blotted onto nitrocellulose sheets. The Coomassie Blue stain was transferred simultaneously and bound strongly to the nitrocellulose, giving a copy of the original gel pattern. Using specific antisera and a second antibody coupled to horseradish peroxidase, proteins in the original mixture could be detected on the nitrocellulose in quantities less than 25 ng. The Coomassie Blue and horseradish peroxidase stains were distinguishable by a suitable filter, allowing precise correlation of spots stained by Coomassie Blue with spots stained with the brown immunoperoxidase reaction product. The technique was used to identify related proteins in electrophoretograms of brain extracts from different animals.
    Additional Material: 4 Ill.
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  • 110
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 48-53 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: High resolution two-dimensional electrophoresis combined with a sensitive silver stain has been used to study the proteins of normal human aqueous humour. The protein patterns of small, unconcentrated, individual samples and of pooled and concentrated aqueous humour samples were compared with plasma or serum from the same individual or with a normal serum. The protein maps of aqueous humour and serum showed great similarity. More than 30 groups of spots were visualized in aqueous humour after silver staining and 18 of them have been tentatively identified. Three polypeptide groups were observed which were not found in plasma or serum. The different clusters, which appeared in the region of high (〉 90 000), medium (50 000-45 000) and low (25 000-15 000) molecular weight polypeptides, remain unidentified. The most abundant cluster represents a molecular weight of approximately 48 000.
    Additional Material: 6 Ill.
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  • 111
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple apparatus and a convenient program for evaluation of two-dimensional electrophoretograms have been developed. The hardware system comprises a charge-coupled device (CCD) camera, four pages of frame memory (FM), a page of display memory (DM), a video-monitor cathode-ray tube (CRT), a digitizer (DIG) and two ports of parallel interfaces (PIO). A small desk-top microcomputer is connected to the apparatus via the interfaces. The system program named frame-imagememory controller (FIMC) has been developed under an assembler of the microcomputer. FIMC is a package of subroutines, and utility programs for two-dimensional densitometry can be written in BASIC language simply with the help of FIMC. Unevenness of basal gray level, caused by the nonuniformity of illumination in the camera's field of view, is eliminated from the pixel data by a method of blank subtraction. Background gray level, caused by protein staining procedures, is corrected by a method of local background subtraction. The two-dimensional densitometry system, equipped with a CCD camera, is applicable not only to transparent gels but also to opaque membranes, and it shows a high reliability in optical measurement in the range of 0-2 O.D.
    Additional Material: 5 Ill.
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  • 112
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 54-55 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Nonspecific antibody binding either to nitrocellulose membrane sites or to transferred and immobilized proteins is eliminated (or minimized to insignificant levels) by treatment with a liquid gelatin preparation. This increases the specificity and sensitivity of nitrocellulose binding immunoassays, and allows incubation at 4°C which minimizes bacterial contamination and/or damage to biologically-labile proteins.
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  • 113
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In a study of intersterility groups in the genus Armillaria, a scanning stage microscope photometer was used to obtain digitized images of photographs of isoelectric focusing patterns. Absorbance values were then mapped from the metric scale to the pH scale. Absorbance values in specified pH intervals were used as features in multivariate statistical procedures to characterize populations and to assign isolates to populations. Digital image analysis techniques were used to obtain an enhanced image of faint or poorly resolved bands.
    Additional Material: 3 Ill.
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  • 114
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 57-57 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 115
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 116
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 59-72 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Additional Material: 5 Ill.
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  • 117
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 73-76 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The electrophoretic separation of RNA species in composite ultra low/medium gelling temperature agarose is described. The resolution obtained was superior to that observed with the standard medium gelling temperature agarose alone. Poly(A)-containing mRNA was successfully fractionated and recovered in a biologically active form from the separating media.
    Additional Material: 4 Ill.
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  • 118
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The method to localize and quantitate glycoproteins on polyacrylamide gels is based on the binding of Concanavalin A to the glycoproteins and the subsequent coupling of horseradish peroxidase to the glycoprotein - Concanavalin A complex. The complex is visualized by the oxidation of 3,3 -diaminobenzidine with horseradish perexidase - H2O2, forming a brownish stain at the gel surface. Various parameters are analyzed and a scheme for the optimal staining of macro and micro slab gels is given. The quantitative evaluation of the stained gels is demonstrated. The staining and quantitation of nitrocellulose transfers is also shown.
    Additional Material: 12 Ill.
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  • 119
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Split products of the third complement factor expressing D but not C epitopes (C3d) were analysed by crossed immunoelectrophoresis using the intermediate gel technique. Four molecular forms of C3d were identified and designated 1, 2, 3 and 4 according to their electrophoretic migration velocity. Whereas the relative concentration of molecular forms 2 and 3 were increased following acute in vivo activation and nephritic factor (NeF)-mediated in vitro activation, the relative concentration of molecular form 1 was found to be increased in patients with chronic activation of the complement system. The results obtained by the addition of anti-C3c antibodies to the first-dimensional gel suggested that form 4 was a C3d-like molecule split from the native C3 during electrophoresis. By the addition of anti-albumin antibodies to the first-dimensional gel it was demonstrated that molecular form 1 of C3d was a complex between C3d and albumin. The immunoprecipitation patterns of molecular forms 2 and 3 was unaffected by anti-albumin and anti-C3c antibodies added to the first-dimensional gel. These data suggest that molecular forms 2 and 3 of C3d are the primary split products of C3b and these molecules are able to form a stable complex with human albumin.
    Additional Material: 4 Ill.
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  • 120
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Alternative procedures for the preparation of wide-range immobilized pH gradients with two-chamber mixers are compared. In the first approach, both vessels contain the same mixture of buffering species, with the addition of either a strong acidic or a strong basic titrant; with the second protocol, the concentration of the buffers is different in the two chambers. The formulations for twenty 2-6 pH unit-wide ranges were computed according to both methods. Relevant parameters of the resulting gradients are listed, including deviation from pH linearity, buffering power, ionic strength and efficiency. The effect of mistakes in the preparation of the gradients is also discussed. “Different concentration” gels allow for a better linearity of the pH course; “same concentration” gels offer a more even buffering power and ionic strength, and assure better reproducibility, in consequence of both the reduced number of manipulations and their higher resistance to mistakes. Reagent consumption and efficiency are very similar in both cases.
    Additional Material: 3 Ill.
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  • 121
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 97-101 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A new electrophoretic system, able to separate all the known standard glycosaminoglycans, was investigated as a method for the study of glycosaminoglycans extracted from tumours of the central nervous system. Densitometric analysis of standard glycosaminoglycan electrophoresis revealed high sesitivity and good repeatability. The electrophoretic system applied to biological samples based on a comigration of extracted glycosaminoglycans with standards, allowed subfractioning of chondroitinsulfates, dermatan sulfate and herapan sulfate and identifaction of heparan sulfate and heparin directly on the sheet by treatment with nitrous acid. Such characters permit the analysis of glycosaminoglycans present in small amount in biological samples.
    Additional Material: 6 Ill.
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  • 122
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Methods for standardization of silver-“stained” protein position in two-dimensional polyacrylamide gel electrophoresis are presented for their application to simultaneous multiple gel systems. Four methods are discussed: the use of a single gel per simultaneous run for standardization with either (1) known quantities of commercially prepared high-or low-molecular-weight standards or (2) a known biological preparation such as T4 phage coat proteins; the use of each gel per simultaneous run to standardize itself by (3) including an internal carbamylation train such as creatine phosphokinase and using a one-dimensional gel pattern of proteins such as rat heart whole homogenate along the margin(s) or (4) identifying “marker” protein spots in test samples that occur in all gels in a given study and standardizing to relative position. Method four is discussed in detail and examples given of its use in a comparative study of proteins in spontaneous primary hepatocellular carcinomas occurring in mice, and its use compared to the other methods.
    Additional Material: 5 Ill.
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  • 123
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Protein migration distances on two-dimensional (2-D) gels were measured in mm (absolute) and in relative internal standard units using creatine kinase carbamylation trains (x-axis) as relative charge (CK-Cr) scale and rat heart extract lines as relative molecular weight (Mr) scale (y-axis). Variation in absolute protein location generally increases with increasing migration distance in both axes with 6-12 mm and 5-11 mm for selected spots in the x- and y-axis respectively; variation in relative migration is significantly less, ranging from 0.9-2.5 mm and 3-4 mm in the x- and y-axis respectively; data are from seven separations run in the same batch, measurements obtained from 1.6 × magnified projections of photographed gels. A computerized plotter was used to generate scaled relative charge - relative molecular weight (Cr-Mr) plots for each separation based upon absolute positions of internal protein standard spots (first dimension) and lines (second dimension). The results support the use of computer-delineated-relative coordinate systems (Cr-Mr plots) for biophysical description of proteins separated by 2-D electrophoresis.
    Additional Material: 6 Ill.
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  • 124
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 122-128 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Alcohol-associated changes in human serum protein patrerns have been detected by silver staining following high resolution two-dimensional electrophoresis and isoelectric focusing-multiple gel piece electrophoresis. In the sera of alcoholics the concentration of α1-acid glycorportein was consistently increased and in a high proporation of sera elevated levels of IgA, α1-antichymotrypsin, haptoglobins and apo A-I lipoprotein were observed. Increased levels of additional unidentified polypeptides were also detected. In contrast, reduced levels of antithrombin III were predominantly associated with the sera of alcoholics. The use of these polypeptides as potential clinical markers for alcohol abuse is discussed.
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  • 125
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A method is presented for the incorporation of [35S]methionine into proteins from freshly microdissected brain tissue samples. Rats were killed by decapitation, the brain was rapidly removed and sectioned without freezing using a vibratome. Hypothalamic nuclei and circumventricular organs of interest were removed by microdissection and incubated in a media containing [35S]methionine for six hours. Proteins within these tissue samples were then separated by two-dimensional polyacrylamide gel electrophoresis. The gels were stained with silver, photographed, dried and exposed to autoradiographic film. The results suggest that 1) this technique will prove useful in examining the uptake of radiolabeled amino acids into proteins from discrete nuclei and other brain areas, and 2) there is considerable regional heterogeneity in the uptake of [35S]methionine by proteins from the different brain regions examined in this study.
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  • 126
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    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 132-132 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 127
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 128
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Defining the properties of antibody preparations has been facilitated by immunoblotting techniques: by ‘double’ immunostaining of blots, however, one can clearly visualize the properties of two (or even more) antibodies on a single blot. We describe a one-day procedure in which two different antisera, both raised in rabbits, can subsequently be applied to a single nitrocellulose blot of protein samples separated by either native or sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. In order to visualize antibody activity, a three-step unlabeled immunoezyme method - the peroxidase anti-peroxidase technique - has been used for both first and second immunostaining. Discrimination between the results of each of the immunostainings has been achieved by using 3,3′ -diaminobenzidine and 4-chloronaphtol as different peroxidase substrates which yield differently colored precipitates. The problem of method overlapping has been overcome by developing a short but efficient elution procedure for the elution of antibody bound to the blot during the first immunostaining: going from ‘mild’ conditions (suited for incubations with sera) to ‘denaturing’ conditions (low pH, low ionic strength, 20 % dimethylformamide) and vice versa repeatedly does not affect antigens but elutes antibodies completely.
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  • 129
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    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 133-138 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two polyacrylamide gel electrophoretic systems for the separation of small peptides are described. In both systems the samples are concentrated by a buffer discontinuity, and the peptides separated according to size and charge in 40-50 % polyacrylamide resolving gels. The acidic gel has an operative pH between 3.0 and 3.1 while the alkaline gel has an operative pH between 9.1 and 9.2. The lower limit of resolution is about 100 daltons for a peptide with one positive charge in the acidic system. The radioactive peptides are detected by autoradiography. Peptides with a free amino group can be fixed in these gels with formaldehyde, so that fluorographic procedures can also be used for increased sensitivity. These techniques enable precise comparison of non-derivatized peptides from complete protein fingerprints and offer a simple one-dimensional analytical method for protein comparison, as well as a general method for separating and analyzing small peptides.
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  • 130
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple and accurate method for the monitoring of the voltage distribution across horizontal slab gels in isoelectric focusing is described. Voltage is measured by passing a single Pt wire electrode (connected to an external voltmeter and to one of the electrodes through the power supply) through holes in the apparatus lid, bored at 5 mm distance from one another across the migration path. Conductances along the migration path calculated from direct voltage measurements are by one order of magnitude lower than those measured by conductimetry on gel slice eluates. Segmental resistances calculated from the measured voltages across adjacent holes in the lid during isoelectric focusing (IEF) add up to the total resistances across the gel obtained from the voltage and current readings on the power supply. In confirmation of previous reports, segmental resistance in IEF was shown to increase dramatically with time, with maxima shifting progressively from pH 4 to 5 in a representative pH3-10 gradient. Joule heat production in IEF decreases with increasing molecular weight of the carrier ampholytes.
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  • 131
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    Weinheim : Wiley-Blackwell
    Electrophoresis 5 (1984), S. 143-147 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A program has been developed for the computer-controlled cooperation of 4 commercially available stepmotor-driven burettes to form density gradients in polyacrylamide gels and solute gradients for other purposes such as centrifugation and low pressure chromatography. The slope of the gradients was defined by entering up to 200 endpoints of straight lines with the percentage of the total volume and the concentration of the gradient-forming solute as coordinates. The possible minimum increment of the discontinuous gradients is 0.5 % of the total range. They are mixed at variable flow rate by two burettes containing the high and low density acrylamide solutions and two additional burettes for the admixture of two catalysts at variable volume proportions. Within 10 min gradients with total volumes between 4 and 650 ml can be delivered. Gradient volumes smaller than 4 ml are possible if an average increment of 〉 0.5 % is accepted. The program meets the requirements of reproducibility for high resolution gradient gel electrophoresis and isoelectric focusing in immobilized pH gradients. All input data for the definition of the gradients can be displayed as a hard copy by a matrix printer and stored on tape. Thus the device meets the requirements of proper documentation of a given gradient in a publication and for reproduction in another laboratory. Other types of gradients for volumes of up to 1.4 liters can be produced by a subprogram converting the 4 burettes into a gradient pump with continuous outflow as used for column chromatography. The speed is variable between 0.4 ml and 5.2 liters per hour with minor pulsation only in the very low range.
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  • 132
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    Electrophoresis 5 (1984), S. 148-154 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The electrophoretic mobility of brush border membrane vesicles purified from the rabbit ileum by chromatography on porous glass beads has been measured with a Laser-Zee microelectrophoresis instrument. The value obtained was measured in a solution containing 0.1 M NaCl, 10 mM HEPES-Tris and 50 mM mannitol at pH 7.37 and 20 °C was -1.41 μm s-1 V-1 cm. The mobility depended on both pH and ionic strength of the solution. The isoelectric point was observed to be at pH 4.25. The brush border membrane vesicle surface was found to be highly negatively charged: there are 1000-2000 negative charges on each vesicle at ionic strength 0.1. Treatments with cation had an effect on mobility and gave a reversal of surface charge.
    Additional Material: 11 Ill.
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  • 133
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    Electrophoresis 5 (1984), S. 155-159 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Human mononuclear cells were split into platelets, monocytes and small lymphocytes by countercurrent centrifugal elutriation (CCE). Determination of the electrophoretic mobilities (EM) of each population revealed that total small lymphocytes, i. e. B, T and null cells, have an equal EM at 1.09 × 10-4 (cm2 V-1 s-1), whereas monocytes showed a symmetric distribution curve with a peak at 0.95 × 10-4 (cm2 V-1 s-1). Thus the cathodal shoulder of the EM distribution curve of total mononuclear cells is caused only by monocytes. Mononuclear cells were stimulated by concanavalin A. Growing and resting lymphocytes were isolated at different times by CCE and analysed by cell electrophoresis. In contrast to murine T cells, human Tlymphocytes did not change their EM within 3 days after mitogenic transformation.
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  • 134
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Phytohemagglutinin (PHA)-the lectin extracted from Phaseolus vulgaris-is found to induce electrophoretic mobility changes of purple membrane particles. The effect is dose-dependent. The ionic strength of the suspending medium modifies the reduction of the membrane surface charge density.
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  • 135
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    Electrophoresis 5 (1984), S. 165-167 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from fresh human bloodstains by ultrathin-layer polyacrylamide gel isoelectric focusing. Electrofocusing of phosphoglucomutase on 0.2 mm thick gels took 52 and 80 min on the Cold Focus Apparatus and Ultrophor, respectively, when the distance separating the electrodes was 8.0 cm, whereas a 9.5 cm distance between electrode wicks required 95 min on the Multiphor to complete isoelectric focusing. The gels with 8.0 cm between electrode wicks provided sharper band patterns compared with the gels with 9.5 cm between electrode wicks.
    Additional Material: 4 Ill.
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  • 136
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A preliminary study has been made of the use of immobilised pH gradients (Immobiline gels) for the separation of the six commonly occurring erythrocyte acid phosphatase (ACP1) phenotypes in blood lysates and stains. These pH gradients are immobilised in the matrix of the polyacrylamide gel and possess different properties from conventional isoelectric focusing (IEF) gels. A comparison was made between the profile of the ACP1 isoenzymes apparent on conventional IEF gels with that seen on the Immobiline gels.
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  • 137
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Fish muscle proteins were separated by slab gel electrophoresis in 10-20 % polyacrylamide gradient gels in presence of sodium dodecyl sulfate. Three muscle extracts were tested: whole homogenate, sarcoplasmic and myofibrillar. Based on differences of the patterns in the 14 000-205 000 molecular weight range, identification of species of different orders, families and genus seems feasible. The most reproducible patterns were obtained with the whole homogenate. No changes were detected in the patterns after cold storage for several days (without freezing).
    Additional Material: 4 Ill.
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  • 138
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    Electrophoresis 5 (1984), S. 171-171 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The mobility of the “Tris-borate” ion at different temperatures and ionic strengths is calculated using previously published equations for calculating the approximate mobility of composed ions.
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  • 139
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    Electrophoresis 5 (1984), S. 172-173 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An ethidium bromide titration procedure using electrophoresis in horizontal multigels is described which facilitates measurement of DNA superhelix densities. Using this system, measured superhelix density was found to be dependent on the amount of DNA loaded on the gel.
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  • 140
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    Electrophoresis 5 (1984), S. 174-175 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A silver staining method is described for increasing the sensitivity of protein detection in agarose gels. Following electrophoresis, the gels are first stained with Coomassie Brilliant Blue R-250, then washed with glutaraldehyde and finally silver stained. Using this double-stain method, protein detection was 100 times more sensitive than Coomassie Blue staining alone and 10 times more sensitive than when silver staining was used alone.
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  • 141
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    Electrophoresis 5 (1984), S. 178-179 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: An improved apparatus for the electrophoretic transfer of proteins from gels to immobilising matrices is described. This device overcomes problems common to previous designs such as entrapment of gas bubbles within the gel sandwich during its assembly and from electrolytic gas generation during transfer. The apparatus has some novel features which facilitate its ease of use and enable several gels to be blotted at one time.
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  • 142
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    Electrophoresis 5 (1984), S. 176-178 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Acrylic acid, methacrylic acid, itaconic acid and dimethylaminopropylmethacrylamide can easily be introduced in polyacrylamide gels in order to obtain reproducible immobilized pH gradients in the pH range 4-6. These acrylic derivatives are commercially available at a relatively low price.
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  • 143
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    Electrophoresis 5 (1984), S. 183-183 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 144
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Proteins exhibit higher apparent molecular weights when treated with mercaptoethanol and separated electropretically in a porosity gradient in presence of ureacompared to their apparent molecular weights without mercaptoethanol. This unexpected effect was first noticed with proteins of jojoba. The apparent increase was also seen when oligomers of serum albumin were separated, but was absent with gelatin, indicating a reaction of disulfide groups in the polypeptide chain of serum albumin. When the proteins were treated with hydrogen peroxide prior to electrophoresis the apparent increase of molecular weights of jojoba proteins after mercaptoethanol treatment was less evident in the presence of monomethylurea and was absent in gels containing tetramethylurea.
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  • 145
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    Electrophoresis 5 (1984), S. 185-185 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 146
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    Electrophoresis 5 (1984), S. 184-184 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 147
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    Electrophoresis 5 (1984) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 148
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    Electrophoresis 5 (1984), S. 187-195 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 149
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Immobilized pH gradients covering, in presence of 8 Murea, the intervals pH 4-7, pH 4-8 and pH 4-9 were applied tothe analysis of human serum. Both formulations with constant buffering power and with constant ionic strengthd were tried. Results are presented of the isoelectric focusing step and of a further fractionation of the sample by sodium dodecyl sulfate- polyacrylamide gel electrophoresis in a two-dimensional by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a two-dimensional system.
    Additional Material: 9 Ill.
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  • 150
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In crossed affinoimmunoelectrophoresis with free concanavalin A (Con A) in the first dimension, four orosomucoid components are normally found in human serum. In this study an optimal range of Con A concentration in the first-dimension gel was defined. The most retarded, fourth component of orosomucoid was shown to be a result of entrapment of Con A-binding molecules in the affinity precipitate. The affinities of the second, weakly retarded, component and of the third, strongly retarded, component to a Con A binding site were found to be identical. The mobilities in the first-dimension electrophoresis of the orosomucoid/Con A complexes were found to be significantly different from each other. Compared to the mobility of the nonretarded, first, component of orosomucoid, the second component had a mobility of 40% and the third component had a mobility of 10%. Differences in the antennary carbohydrate structures on the glycosylation sites of orosomucoid are suggested as an explanation for these observations.
    Additional Material: 4 Ill.
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  • 151
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Using panicum mosaic virus (PMV) and several St. Augustine decline strains of PMV (SAD) as test viruses, previously developed procedures of agarose gel electrophoresis (reviewed in P. Serwer, Electrophoresis 1983, 3, 375-382) have been used for the first time to detect and characterize plant viruses. PMV and some SAD strains produce two particles that copurify during rate zonal sedimentation, but form separate bands during agarose gel electrophoresis (forms 1 and 2). By measuring electrophoretic mobility (μ) as a function of agarose percentage and correcting for electroosmosis, it was found that: (a) form 1 has a solid support-free μ (μ0) different from the μ0 of form 2, and (b) forms 1 and 2 have the same radius (14.6 ± 0.6nm). Form 1 is indistinguishable from form 2 by either electron microscopy or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The μ0 of form 1 and the relative amount of forms 1 and 2 vary with the virus strain and can be used to help identify strains. A previously described satellite of PMV and some SAD strains also has two forms. The radius of the satellite of one SAD strain was found to be 7.6 ± 0.3 nm. A procedure of differential gel staining was developed for distinguishing the 14.6 nm virus from its 7.6 nm satellite. After agarose gel electrophoresis of either totally unfractionated or partially fractionated, infected grass extracts, virus particles wer detected without interference from host components. The procedures used here should be useful for rapid disease diagnosis and strain typing of the above and possibly other plant viruses.
    Additional Material: 5 Ill.
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  • 152
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Albumin and glycosylated albumin from serum and urine of 10 diabetic patients have been studied by ultrathin-layer isoelectric focusing andsilver staining. Both total serum albumin and its glycosylated form were microheterogeneous with a main band focused in the pH 4-5 range; in the case of glycosylated ablumin several cathodic bands with pI's up to 7 were found to form a discretely abundant population among the total species. Some similarites and some striking differences were observed for urinary albumin, which, beside the main unmodified albumin with pI ca. 4.8, was enriched in microheteregeneous more anodal bands (PI 4.0-4.7 species). All these anodal bands were tightly bound by concanavalin A-Sepharose, suggesting their identity as glycosylatedalbumin. these observations indicate that the glycosylation of albumin is not an equimolar reaction, but it can involve many sites of attachement of carbohydrates. The also suggest that albumin species with higher net negative charge and higher content of carbohydrates are preferentially able to pass through the glomerular barrier, providing the descriptive basis of diabetic albuminuria. Their relevance to diabetic nephropathy is a goal for further work.
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  • 153
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Proteins from cerebrospinal fluid (CSF) were separated by two-dimensional electrophoresis (2-DE), visualized with silver staining and quantitated with computer-assisted densitometry. This report describes the polypeptide variations observed and their correlations to disease states. CSF from 22 multiple sclerosis (MS) patients was compared to CSF from: 20 non-inflammatory neurological disease cases (NINDC), 17 inflammatory neurological disease cases (INDC) including a sub-set of 6 patients with sub-acute sclerosing panencephalitis (SSPE) and 14 normal volunteers. In a specific comparison between the MS group and the normal volunteers, 32% of the polypeptides varied quantitatively (p 〈 0.05 or greater). The NINDC group resembled the normal volunteers, while the INDC and SSPE groups resembled the MS group. In general, the quantitative changes in the polypeptides analysed in this study permit one to distinguish one disease state from another. In addition to these quantitative alterations, we also report the appearance of additional immunoglobulin light chain species in MS, SSPE, Herpes simplex encephalitis, neurosyphilis, one patient with Shy-Drager syndrome and one patient with idiopathic orthostatic hypotension. No other disease-specific qualitative changes were observed in the form of either deletions of proteins normally present, or additional proteins. This survey utilizing 2-DE has demonstrated protein alterations in the CSF of patients with varied neurological diseases compared with normal CSF, which may prove useful in clinical diagnosis.
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  • 154
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    Electrophoresis 5 (1984), S. 251-251 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Silanization of plastic foils or glass plates is necessary for fixation of polyacrylamide gels to the support. When using glass plates as support, silanization creates problems which can be completely avoided when the silanized plates are heated for 30-45 min at 55-60°C prior to gel polymerization.
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  • 155
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    Electrophoresis 5 (1984), S. 245-250 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The proteins of human whole saliva have been detected by either silver or Coomassie Brilliant Blue R-250 staining following two-dimensional electrophoresis using either protein denaturing or non-denaturing conditions. The high resolution protein denaturing technique revealed over 600 polypeptides and the non-denaturing technique over 200 proteins in 50 μl of unconcentrated saliva. Silver staining enhances (50-100 fold) detection of the majority of these salivary components and reveals (as clear spots) both polypeptide and proteins not detected with Coomassie Brilliant Blue R-250. Nevertheless, optimal silver staining of proteins (non-denaturing conditions) requires pre-equilibration of the gels in aqueous sodium dodecyl sulphate. In contrast, Coomassie Brilliant Blue R-250 reveals diffusely staining pink zones not detected by silver staining. The possible nature of salivary components demonstrating unusual staining characteristics is discussed.
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  • 156
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    Electrophoresis 5 (1984), S. 269-274 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The changes of the electrophoretic mobility (EM) of human monocytes during invitro maturation into macrophages were investigated. Incubation of isolated monocytes for longer than 4 days was always accompanied by an increase of the EM of the monocytes. This increase of the EM could only be inhibited when lymphocytes were incubated together with the monocytes. The inhibitory effect of the lymphocytes was optimal when the culture medium was supplemented by autologous plasma and when the cell suspension was kept in teflon beakers, but it was reduced when the culture medium was supplemented by fetal calf serum (FCS) and when the cell suspension was kept in polystyrene dishes. The macrophages, which showed increased EM after 10 days of incubation, had equal antibody-dependent cell-mediated cytotoxic (ADCC) activity but lower plaque-forming activity than the macrophages which retained their original EM during incubation. The finding of macrophage sub-populations which differ in their EM provides a good opportunity to isolate and further investigate macrophages, which may have different biological activities.
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  • 157
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A number of different carrier ampholyte mixtures were compared in order to produce a pH gradient which separated the phosphoglucomutase-1 (PGM1) phenotypes most efficiently. Ultrathin gels incorporating a mixture of pH 5-7 and pH 6-8 Ampholine was shown to produce the best results. Application of samples to the cathodal end of the gel reduced the problem of overloading which is frequently encountered with ultrathin gels.
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  • 158
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    Electrophoresis 5 (1984), S. 263-269 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Film detection of compounds labeled with the weak beta-emitters 3H and 14C is demonstrated for electroimmunoprecipitated proteins in agarose gels by means of the water-soluble fluorophor sodium salicylate. The degree of enhancement of film detection of tritium and carbon-14 with 0.7 M sodium salicylate paralleled fluorography with diphenyloxazole on polyacrylamide gels. The method was optimized and the sensitivity, expressed as radioactivity per cm immunoprecipitate, was 0.9 nCi/cm (2000 dpm/cm) in 24 h for 3H and 0.4 nCi/cm (830 dpm/cm) in 18 h for 14C. The procedure was found superior to the commercially available enhancement solutions ‘EnHance’ and ‘Amplify’. Salicylate fluorography on Kodak X-Omat L and AR-5 films was about 30 and 60 times more sensitive than Ultrofilm 3H. With 14C-labeled anti-antibodies rocket electroimmunoprecipitation corresponding to about 50-100 pg protein could be visualized in 7 days. Conclusively, the method is rapid (soaking and drying require about 30 min), sensitive, reproducible, cheap, and advantageous if the immunoplates are to be stained for protein after fluorography.
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  • 159
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    Electrophoresis 5 (1984), S. 354-357 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We describe the use of 35S-labelled stains for the detection of submicrogram amounts of protein in SDS polyacrylamide gels. Many stains used to detect protein can be rendered radioactive, and proteins can be detected by radiofluorography due to stain binding in the absence of visible color. Of the stains tested. [35S]Ponceau Red S and [35S]Serva Blue R gave the best results. The potential utility of protein detection with radioactive stains is discussed.
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  • 160
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    Electrophoresis 5 (1984), S. 358-361 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Dansylated proteins have been used in combination as isoelectric point and molecular weight standards and included in gel samples for separation by two-dimensional polyacrylamide gel electrophoresis. These proteins provide invariant points of reference that facilitate the unequivocal identification of proteins separated by two dimensional polyacrylamide gel electrophoresis without affecting resolution. Moreover, immunoautoradiograms of Western blots can be aligned with their corresponding gel patterns with great accuracy. The logical extension of the established usage of dansylated protein standards to these applications is described.
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  • 161
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    Electrophoresis 5 (1984), S. 370-372 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A technique for the preparation of ultrathin titration curve gels, 50 μm polyacrylamide gels and 100 μm agarose gels, is presented. By separating protein standard mixtures the advantages of this ultrathin-layer technique in terms of more convenient handling, cost and time are demonstrated.
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  • 162
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    Cell Biochemistry and Function 2 (1984), S. 63-63 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 163
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    Cell Biochemistry and Function 2 (1984), S. 69-70 
    ISSN: 0263-6484
    Keywords: Liver ; thyroglobulin ; lysosomes ; intralysosomal hydrolysis ; thyroid ; TSH ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In vitro, poorly iodinated thyroglobulin (Tg) is hydrolysed at the same rate whether it is enclosed in thyroid or in liver lysosomes, while fully iodinated Tg is degraded faster inside liver lysosomes. After in vivo TSH administration, thyroid lysosomes hydrolyse fully iodinated Tg as fast as do liver lysosomes.
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  • 164
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    Cell Biochemistry and Function 2 (1984), S. 89-94 
    ISSN: 0263-6484
    Keywords: Immunology ; cytotoxic T cells ; lysosomes ; quantitative cytochemistry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The mechanism of the lysis of target cells by cytotoxic T-cells (Tc) is still obscure; there is no evidence for transfer of material from the Tc and prior to lysis, despite intimate contact, the plasma membranes of both types of cell appear to remain intact. The effects on the target cell lysosomes of brief contact between anti-viral Tc and targets bearing both the appropriate histocompatibility and viral antigens, have been examined cytochemically. Both the distribution of acid phosphatase activity and the percentage bound lysosomal naphthylamidase activity indicated that, in virus-infected target cells exposed to Tc, the lysosomal membranes became totally labilized. Thus the contact between Tc and targets appears to cause sufficient perturbation of the target plasma membrane as to cause the intracellular release of some agent that activates ‘suicide capsule’ lysosomes.
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  • 165
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    Cell Biochemistry and Function 2 (1984), S. 127-128 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 166
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    Keywords: Fats ; adipose tissue ; cold adaptation ; mitochondria ; creatine kinase ; peroxisomes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.
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  • 167
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    Cell Biochemistry and Function 2 (1984), S. 10-11 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 168
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    Cell Biochemistry and Function 2 (1984), S. 23-25 
    ISSN: 0263-6484
    Keywords: Blood ; fatty acids ; platelet ; membrane ; myeloproliferative disorders ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The fatty acid composition of platelet membranes has been analysed in patients with thrombocytosis due to myeloproliferative disorders, who had not taken any drugs. A significant increase in palmitic and oleic acid, together with a decrease in stearic, linoleic and arachidonic acids was observed. The fatty acid pattern of platelet membranes was also analysed in patients during treatment with ASA (acetylsalicylic acid). ASA ingestion completely normalizes the platelet content of palmitic acid and partially that of stearic and arachidonic acid, whereas it has no effect on the level of linoleic acid and raises that of oleic acid. The altered pattern of fatty acids observed in patients may interfere with platelet function by decreasing membrane fluidity. Treatment of patients with ASA seems to act on platelet membranes by partially normalizing the fatty acid composition.
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  • 169
    ISSN: 0263-6484
    Keywords: Carbohydrates ; membrane ; glycosylation ; retrovirus ; cytotoxicity ; lymphoma ; metastasis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In order to correlate the biochemistry of cell surface carbohydrates with cell function, we have treated cells with swainsonine and followed the biochemical and functional modifications induced by this compound. After treatment with swainsonine, surface glycoproteins had a lower apparent molecular weight and a higher isoelectric point. This is compatible with the replacement of complex carbohydrates by hybrid or high-mannose carbohydrates. Several functional tests were unaffected. However, swainsonine-treated cells displayed an altered pattern of in vivo homing, suggesting that carbohydrates play a role in this process.
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  • 170
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    Cell Biochemistry and Function 2 (1984), S. 153-154 
    ISSN: 0263-6484
    Keywords: Tissue biology ; immunofluorescent staining ; monoclonal antibodies ; S-phase cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This note describes an immunofluorescent staining method for cells in the S-phase which have been allowed to take up bromodeoxyuridine into their DNA in place of thymine. The technique involves the use of fluorescinated monoclonal antibodies against bromodeoxyuridine and allows rapid and accurate estimation of cells in the S-phase, the technique does not require interpretation by skilled technicians.
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  • 171
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    Cell Biochemistry and Function 2 (1984) 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 172
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    Cell Biochemistry and Function 2 (1984), S. 115-118 
    ISSN: 0263-6484
    Keywords: Protozoa ; Entamoeba ; polyamines ; h.p.l.c. ; DFMO ; ODC ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The polyamine content of Entamoeba was measured by a procedure that involved benzoylation followed by high performance liquid chromatography (h.p.l.c.). A high concentration of putrescine and significant amounts of spermidine and spermine were found in actively growing trophozoites and in the cyst forms of the organism. In contrast, trophozoites in stationary phase had greatly reduced amounts of putrescine and exhibited peaks in h.p.l.c., possibly indicative of acetylated polyamines. α-D,L-difluoromethylornithine (DFMO) lowered the concentration of polyamines in growing trophozoites, but did not inhibit the degree of proliferation. There is evidence for pathways of polyamine biosynthesis in Entamoeba other than through ornithine decarboxylase (ODC).
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  • 173
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    Cell Biochemistry and Function 2 (1984), S. 6-10 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 174
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    Cell Biochemistry and Function 2 (1984), S. 21-22 
    ISSN: 0263-6484
    Keywords: Blood ; erythrocyte membrane ; 99Mo labelling ; spectrin subunits ; specific binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Molybdenum in the form of its pentavalent complex binds primarily to spectrin when incubated with erythrocytes. Only the band 1 subunit is involved in this interaction thus indicating some structural differences between spectrin subunits.
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  • 175
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    Cell Biochemistry and Function 2 (1984), S. 38-42 
    ISSN: 0263-6484
    Keywords: Cytology ; ascites cells ; endoplasmic reticulum ; subfraction ; cytochalasin B ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Information on the interaction between endoplasmic reticulum (ER) membranes and components of the skeletal network of the cell was gained by treating cells with the antimicrofilament agent cytochalasin B prior to cell disruption by nitrogen cavitation. Treatment of Krebs II ascites cells with cytochalasin B (5-10 μg ml-1) resulted in an increased yield of three ER membrane subfractions  -  heavy rough (HR), light rough (LR) and smooth (S) membranes, as judged by 3H-choline incorporation in gradient fractions following discontinuous sucrose gradient centrifugation. The major increase was observed in the HR fraction. These results indicate that the actual yield of the respective ER membrane subfractions after cell disruption is dependent on the degree of direct and/or indirect interaction between individual ER membranes and actin containing filaments of the cytoskeleton in the intact cell.
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  • 176
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    Cell Biochemistry and Function 2 (1984), S. 43-48 
    ISSN: 0263-6484
    Keywords: Dental cement ; cytotoxicity ; in vitro ; glass ionomer cements ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The new glass ionomer dental cement, ChemFil, was found to be moderately cytotoxic when freshly mixed in vitro. This was assessed by a reduction in fibroblast and macrophage counts following 24 h exposure to the material, and by alterations in enzyme levels and enzyme staining kinetics relative to control cultures. Testing of set samples of the material showed that its toxicity decreased rapidly with setting. ChemFil behaved in a manner similar to two older glass ionomers, ASPA and ChemBond, although it was generally slightly less irritant.
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  • 177
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    Cell Biochemistry and Function 2 (1984), S. 57-61 
    ISSN: 0263-6484
    Keywords: Heart ; myocardial biopsies ; cryostat sections ; interference microscope ; densitometric trace ; oedema ischaemic arrest ; reperfusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Oedema following periods of ischaemic arrest and subsequent reperfusion has been shown experimentally and clinically to affect the functional state of the heart. Tissue water content has been measured in myocardial sections by microscopic interferometry and densitometry, and the results correlated with those obtained by wet and dry weight analysis (r = 0.87; p 〈 0.001). Microscopic interferometry also revealed the distribution of the water in the tissue. Experimentally induced ischaemic arrest in isolated rat hearts resulted in predominantly intra-fibrillar oedema, whilst subsequent reperfusion resulted in interfibrillar oedema. Microscopic interferometry facilitates accurate measurement of water content in tissue samples as small as 2 mg wet weight and shows (as conventional wet/dry weight analysis cannot) the distribution of the water in the tissue.
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  • 178
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    Cell Biochemistry and Function 2 (1984), S. 67-67 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 179
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    Cell Biochemistry and Function 2 (1984), S. 85-88 
    ISSN: 0263-6484
    Keywords: Liver ; biomembranes ; liver microsome ; fluorescence ; 5′-nucleotidase ; silymarin ; silybin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Silymarin and silibyn are extracted from the seeds of Silybum marianum and used as a liver protectant because of their free radical scavenging. When incorporated into rabbit liver microsomes they cause a small decrease in the flourescence anisotropy of 1,6-diphenyl-1,3,5-hexatrine (DPH) but not of 1-anilinononaphthalene-8-sulphonic acid (ANS), incorporated into the membranes. They do, however, reduce the fluorescence intensity of incorporated ANS without changing the wavelength of maximum intensity. These observations suggest that the drugs are incorporated into the hydrophobic-hydrophilic interface of the microsomal bilayer and perturb the structure by influencing the packing of the acyl chains.
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  • 180
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    Cell Biochemistry and Function 2 (1984) 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 181
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    Cell Biochemistry and Function 2 (1984), S. 119-124 
    ISSN: 0263-6484
    Keywords: Bones ; bone marrow ; cAMP ; cGMP ; erythropoietin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.
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  • 182
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    Cell Biochemistry and Function 2 (1984), S. 140-144 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 183
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    Cell Biochemistry and Function 2 (1984), S. 144-148 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 184
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    Cell Biochemistry and Function 2 (1984), S. 167-170 
    ISSN: 0263-6484
    Keywords: Blood ; avian thrombocytes ; platelet ; adenine nucleotides ; malonyldialdehyde ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Analysis of free nucleotide composition of both avian thrombocytes and pig platelets showed quantitative differences in the level of adenine nucleotides. 3H-adenine taken up by turkey thrombocytes was metabolized mainly to adenine nucleotides was not released after thrombin action. Thrombin liberated non-radioactive adenine nucleotides (18.2 ± 1.5 %, 20.6 ± 1.9%) of the total, probably localized in a storage pool. Malonyldialdehyhyde (MDA) production due to thrombin was observed in both platelets and thrombocytes.
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  • 185
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    Cell Biochemistry and Function 2 (1984), S. 184-184 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 186
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    Cell Biochemistry and Function 2 (1984), S. 171-176 
    ISSN: 0263-6484
    Keywords: Blood ; human erythrocyte ; erythrocyte membrane ; choline phospholipid ; acyl chain ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Intact human erythrocytes were treated, under non-haemolytic conditions at 37°C, with synthetic phosphatidylcholine which has homologous, saturated acyl chains of 8 ∼ 18 even-numbered carbon atoms (C8 ∼ C18-PC) or with lysophosphatidylcholine which has a saturated acyl chain of 8 ∼ 18 carbon atoms (C8 ∼ C18-lysoPC). The C8 ∼ C14-PC and C12 ∼ C18-lysoPC species were rapidly incorporated into the erythrocytes and induced a shape change of the crenation (echinocyte formation) type. The site of the incorporation was found to be most probably on the outer leaflet of the membrane lipid bilayer. The extent of the shape change was dependent on the amount of each lipid incorporated. When the same amount of a PC or lysoPC species was incorporated into the membrane, about the same extent of crenation was induced, independent of acyl chain length. However, C16-PC, C18-PC, C8-lysoPC and C10-lysoPC, which were not incorporated into the erythrocytes, did not induce any shape change. It is therefore suggested that the hydrophobic moiety of these amphiphilic lipids may greatly contribute to their transfer from the outer medium into the erythrocyte membrane, but do not influence so much the perturbation of the membrane lipid bilayer which may be responsible for induction of the shape change.
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  • 187
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    Cell Biochemistry and Function 2 (1984), S. 186-194 
    ISSN: 0263-6484
    Keywords: Iron ; ferritin ; haemosiderin ; lysosomes ; haemochromatosis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: High levels of haemosiderin occur in iron overload syndromes such as idiopathic haemochromatosis or secondary iron overload in thalassaemic patients; haemosiderin is the predominant iron-storage compound in such cases. It consists of a large aggregate of FeOOH cores, many of which have an incomplete shell of protein, and is probably derived from ferritin by lysosomal proteolysis. In addition, some chemical degradation of the ferritin cores appears to occur on conversion to haemosiderin. Other biochemical components are phosphate and magnesium, which may be adsorbed to the core surface, and perhaps certain lipids. Haemosiderin may have a central role, either directly or indirectly, in iron cytotoxicity and therefore the chemistry and biochemistry of this material warrants further study.
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  • 188
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    Cell Biochemistry and Function 2 (1984), S. 257-262 
    ISSN: 0263-6484
    Keywords: Blood ; 2,3-bisphophoglycerate ; inositol-5-phosphate ; 2,3-bisphophoglycerate synthase ; phytase ; counter current distribution ; avian red cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Two generations of red cells (embryonic and definitive), different types of haemoglobins, and special organic phosphates involved in the control of haemoglobin oxygenation (2:3-bisphosphoglycerate, BPG, and inositol-5-phosphate, IP5), have been found progressively during development of the chick. Levels of both organic phosphates, as well as activities of the enzymes involved in BPG synthesis (2:3-bisphosphoglycerate synthase, BPGM) and IP5 formation (phytase), were studied in the erythrocyte populations from embryo, young and adult chickens. Measurement of specific activities of BPGM and phytase in the two subpopulations present in young chickens showed that these phosphates could be specifically and predominantly formed in these two red cell populations.
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  • 189
    ISSN: 0263-6484
    Keywords: Metabolism ; established cell cultures ; hepatoma cells ; drug metabolism ; cytochrome P450 ; inducers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A cell line derived from a Morris hepatoma, MH1C1, was examined for its in vitro expression of monooxygenases. These cells were found to contain different forms of cytochrome P450, as shown by the response to inducers, namely phenobarbital (PB), 3-methylcholanthrene (MC) and metyrapone (MP). MH1C1 cell monolayers exposed to PB or MC showed an increase in the concentration of two spectrally distinct forms of cytochrome P450. The PB and MC treatments elicited enzyme activities towards the substrates aminopyrine and benzo(a)pyrene, respectively. The cell treatment with metyrapone led to a simultaneous stimulation of aminopyrine demethylase and benzo(a)pyrene hydroxylase activities, so underlining the peculiar features of this inducer.
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  • 190
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    Cell Biochemistry and Function 2 (1984), S. 78-84 
    ISSN: 0263-6484
    Keywords: Liver ; erythrocyte ; chromatin ; nuclease ; non-histone protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Chicken liver and erythrocyte nuclei were separated by mild treatment with micrococcal nuclease into nuclease sensitive (NS) and nuclease resistant (NR) fractions, differing in chemical composition and transcriptional activity in vitro. Nuclei, NS and NR fractions of both tissues were fractionated by hydroxyapatite chromatography into three groups of non-histone chromatin proteins (NHCP) and characterized by SDS-polyacrylamide gel electrophoresis. Some differences in the molecular distribution of non-histone proteins of chicken liver and erythrocytes between nuclease sensitive and resistant parts of chromatin have been described.
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  • 191
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    Cell Biochemistry and Function 2 (1984), S. 71-77 
    ISSN: 0263-6484
    Keywords: Liver ; taurine transport ; rat hepatocytes ; primary culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2°C was unsaturable, whereas that at 37°C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 μM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37°C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.
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  • 192
    ISSN: 0263-6484
    Keywords: Intestine ; epithelial cells ; rat ; electron microscopy ; O-deethylation ; glucuronidation ; 7-ethoxycoumarin ; 1-naphthol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Rat intestinal epithelial cells were isolated by EDTA-chelation, combined with gentle shaking (modified Weiser procedure) or with strong longitudinal vibration (Harrison/Webster procedure). Both methods yield large numbers of viable cells and are relatively easy to use. Electronmicroscopical and biochemical data indicate that cell fractions from different levels of the villous region can be obtained only by the modified Weiser procedure. When strong mechanical forces are involved (Harrison-Webster procedure) the villus epithelium is released according to an all-or-nothing process. The biotransformational capacity of cell fractions, obtained from different levels of the villi by the modified Weiser procedure, was investigated. It was shown that the rate of metabolism of 7-ethoxycoumarin and 1-naphthol was substantially higher in lower villous cells than in cells isolated from the upper villous region. O-Deethylation of 7-ethoxycoumarin decreases from 145 ± 13 pmole/min mg cell protein (72 ± 4% conjugated) in lower villous cells to 62 ± 12 pmole/min mg cell protein (37 ± 6% conjugated) in tip cells. Glucuronidation of 1-naphthol decreased from 495 ± 23 pmole/min mg cell protein (lower villous cells) to 137 ± 13 pmole/min mg cell protein (tip cells).
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  • 193
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    Cell Biochemistry and Function 2 (1984), S. 107-110 
    ISSN: 0263-6484
    Keywords: Hormones ; thyroid hormones ; Na+,K+-ATPase ; renal medulla ; cytochemical bioassay ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+, K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+, K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+, K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 - 1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.
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  • 194
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    Cell Biochemistry and Function 2 (1984), S. 208-212 
    ISSN: 0263-6484
    Keywords: Hormones ; GnRH ; receptor ; subcellular distribution ; positive cooperativity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 ± 0.07, 0.78 ± 0.04, 0.31 ± 0.03 and 0.27 ± 0.03 fmol μg-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10-9 M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p 〉 0.5) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p 〈 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p 〈 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells. A single, low-capacity binding site may, or may not suggest the presence of a structurally incomplete form of the receptor protein in microsomes and nuclei. Binding to the secretory granules fraction exhibited only a relatively minor temporal difference compared to the plasma membrane, which may have resulted from an inappropriate conformational state of the receptor protein. Only the binding to the plasma membrane exhibited appropriately both the affinity and temporal requirements of the intact GnRH receptor protein in vitro and in vivo.
    Additional Material: 5 Ill.
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  • 195
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984) 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 196
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984), S. 15-20 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
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  • 197
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984), S. 11-15 
    ISSN: 0263-6484
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
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  • 198
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984), S. 26-32 
    ISSN: 0263-6484
    Keywords: Hormones ; gonadotropins ; Leydig cells ; perfusion ; steroid hydroxylase ; steroid oxidoreductase ; testis ; testosterone ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Production of testosterone by highly purified Leydig cells prepared from rat and mouse testes is compared. Testosterone formation is improved to a higher degree in rat (2.7-fold) than in mouse (1.7-fold) cells by callagenase treatment of the testis compared with mechanical isolation. Mouse Leydig cells respond to exogenous stimuli (chorigonadotropin, dibutyryl cyclic AMP) with 2.4-fold higher testosterone secretion than rat cells. A 1.7-fold increased conversion of androgen precursors to testosterone by mouse compared with rat Leydig cells is demonstrated in static incubations as well as in steady-state superfusion experiments and can be derived from enhanced androstenedione reduction and a less inhibitory effect of progesterone on this process is mouse Leydig cells.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 199
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984), S. 33-37 
    ISSN: 0263-6484
    Keywords: Blood ; lymphocyte ; nude mouse ; DNA ; antibodies ; allotype ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
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  • 200
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 2 (1984), S. 53-56 
    ISSN: 0263-6484
    Keywords: Heart ; heparin ; lipoprotein lipase ; secretin ; cardiocytes ; sulphated glycosaminoglycans ; dexamethasone ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When cardiac muscle cells from mature rats were incubated in vitro in the presence of heparin (8.7 nmole ml-1) lipoprotein lipase activity appeared in the incubation medium. The intracellular activity of the enzyme remained unchanged. Other glycosaminoglycans (heparan sulphate, dermatan sulphate, keratan sulphate and chrondroitin 6-sulphate) at the same or higher concentrations were totally ineffective in producing any enzyme redistribution between cells and medium. The release seen in the presence of heparin was blocked by the presence of cycloheximide. Cycloheximide by contrast had no effect on the release observed in the presence of dexamethasone, The action of endogenous glycosaminoglycans are unlikely therefore to have a significant role to play in the movement of lipoprotein lipase in heart tissue in vivo.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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