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111.
Hereditary heat-labile hexosaminidase B: its implication for recognizing Tay-Sachs genotypes 总被引:2,自引:2,他引:0
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R Navon J Nutman R Kopel L Gaber N Gadoth B Goldman M Nitzan 《American journal of human genetics》1981,33(6):907-915
Two pairs of alleles, at the two loci of hexosaminidase (HEX), were found to segregate in an Arab inbred family: the normal and the mutant Tay-Sachs (TSD) alleles of HEX A, and the normal and a mutant allele of HEX B. Since the mutant HEX B is heat labile, no reliable identification of TSD genotypes can be obtained in its presence, as long as the proportions of HEX A and B are estimated by the routinely used heat-inactivation method. The genotypes may be correctly identified in such cases by separation of the two isoenzymes on ion-exchange chromatography, estimating their individual activities, and calculating the ratio between them. Of the nine genotype combinations possible with these two pairs of alleles, five have been identified in the reported family by this procedure. 相似文献
112.
A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum 总被引:18,自引:9,他引:9
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R C Goldman D White F Orskov I Orskov P D Rick M S Lewis A K Bhattacharjee L Leive 《Journal of bacteriology》1982,151(3):1210-1221
The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum. 相似文献
113.
Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes 总被引:16,自引:0,他引:16
Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors. 相似文献
114.
Selective isolation and culture of a proliferating epithelial cell population from the hamster trachea 总被引:7,自引:0,他引:7
William E. Goldman Joel B. Baseman 《In vitro cellular & developmental biology. Plant》1980,16(4):313-319
Summary A reliable cell isolation technique was developed to allow the cultivation of cells from the hamster respiratory tract. Repeated
thermolysin treatments and gradient centrifugation yielded a cell culture completely free from contamination by fibroblasts.
Viable cells could be isolated from as little tissue as a single hamster trachea, but in vitro proliferation occurred only
if the hamster was less than 4 months of age. The cultured cells could be repeatedly passaged and subcultured for weeks by
employing normal tissue culture techniques. Morphologically, the monolayers appeared to be a homogeneous population of epithelial
cells, and successful cloning of freshly isolated single cells resulted in apparently identical cultures. The epithelial origin
of these cells was also suggested by continued growth in minimum essential medium withd-valine substituted forl-valine. The relative ease with which this cell type can be isolated, cultured, and manipulated in vitro should encourage
its application as a model of the respiratory epithelium.
This research was supported by Public Health Service Grant P50-HL 19171 and Research Career Development Award 1-K04-AI 00178
to J. B. B. 相似文献
115.
Isolation and partial characterization of membrane protein constituents of human neutrophil receptors for chemotactic formylmethionyl peptides 总被引:5,自引:0,他引:5
Plasma membranes of human neutrophils were solubilized in buffer containing a nonionic detergent and applied to a formylmethionylleucylphenylalanine (fMet-Leu-Phe)-Sepharose column that was washed and eluted with the chemotactic peptide fMet-Leu-Phe. Analysis of the eluate by filtration on Bio-Gel P150 in sodium dodecyl sulfate (NaDodSO4) buffer and by NaDodSO4-polyacrylamide gel electrophoresis revealed three predominant membrane proteins of approximate molecular weight 94 000 (MP-1), 68 000 (MP-2), and 40 000 (MP-3), of which MP-2 accounted for 74--93% of the total protein. Purified MP-1 and MP-2 contained an above average content of hydrophobic amino acids, while MP-2 and MP-3 had an above average content of acid and/or amide amino acids and a below average content of basic amino acids. MP-2 and MP-3, but not MP-1, bound [3H]fMet-Leu-Phe in equilibrium dialysis chambers. Both MP-2 and MP-3 exhibited high-affinity sites with a valence of 0.2--0.3 and mean KA values of 9 x 10(8) and 2 x 10(7) M-1, respectively, and low-affinity sites with a valence of 0.3--0.5 and mean KA values of 3 x 10(7) and 2 x 10(6) M-1 (n = 3). The specificity of the binding of fMet-Leu-Phe was suggested by the failure of MP-2 and MP-3 to bind lipid chemotactic factors and to adhere to a Sepaharose column to which had been coupled chemotactic fragments of the fifth component of complement. A series of synthetic formylmethionyl peptides exhibited the same rank order of potency as inhibitors of the binding of [3H]fMet-Leu-Phe by MP-2 and as stimuli of neutrophil chemotaxis. Membrane proteins isolated by fMet-Leu-Phe-Sepharose affinity chromatography may represent constituents of specific human neutrophil receptors for chemotactic peptides. 相似文献
116.
Cells of Bacillus subtilis, when tethered by using antiflagellar antibody, rotate briefly counterclockwise (swimming behavior) or clockwise (tumbling behavior) when amino acids are added or removed, respectively. "Dissociation constants" for attractant-binding site interactions, calculated from duration of the rotational response to addition of amino acids, agreed with those calculated for their removal and with previous values calculated from sensitivity capillary assays. The ratio of adaptation times for addition versus removal of attractant averaged 1.7, which differs greatly from the value of 50 for Escherichia coli. 相似文献
117.
118.
A Ca2+-dependent modulator protein has been isolated from BHK-21 cells. The purification requires heat treatment, ion-exchange chromatography, and gel filtration. The protein appears homogenous on sodium dodecyl sulfate--polyacrylamide and isoelectric focusing gels. The protein comigrates with purified smooth muscle and brain modulators. BHK-21 modulator is characterized by a high content of aspartic and glutamic acids and by a high phenylalanine/tyrosine ratio. It lacks both cysteine and tryptophan. The protein is effective in activating brain-modulator-deficient phosphodiesterase. It can also be used in assay systems to generate Ca2+-sensitive actin activation of both BHK-21 and smooth muscle myosins. Therefore, it is proposed that the BHK-21 modulator protein is a component of the Ca2+-dependent mechanism involved in the regulation of actin--myosin interactions in BHK-21 cells. 相似文献
119.
Liposomes can be separated from low molecular weight solutes on minicolumns of Sephadex G-50 made from the barrels of 1- or 5-ml plastic syringes. Excess fluid is first removed from the Sephadex beads by centrifugation and a mixture of liposomal entrapped and free solute is applied to the column bed. The centrifugation is repeated forcing the liposomal material through the column into a test tube while the free solute is quantitatively retained in the Sephadex. The procedure is applicable to a variety of solutes and 92 to 100% recovery is achieved for both charged and neutral liposomes. This technique has advantages over other methods for separating extraliposomal solutes from liposomes. Numerous samples can be processed simultaneously within minutes with no dilution of the liposomal preparation. Nonentrapped solute within the Sephadex can be easily recovered in a small volume of water or buffer. 相似文献
120.