Biomedical subjects

L M Sorokin

Publications and source records attributed to L M Sorokin.

7 recordsLinked to original sources

PubMed1992

Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development.

Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC50 approximately 1 microgram/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to alpha 6 beta 1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.

Animals↗

PubMed1989

Transformation-induced changes in transferrin and iron metabolism in myogenic cells.

The uptake of transferrin and iron by cultured myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus (tsLA24) was compared with that of normal developing myogenic cells which were proliferating at the same rate as the transformed cells. The mechanism of transferrin and iron uptake was the same in the transformed cells as in normal myogenic cells and involved receptor-mediated endocytosis of transferrin. However, there were differences in transferrin receptor numbers and receptor function. The number of receptors in transformed cells was more than twice as great as in the normal cells largely due to increased surface receptor numbers. Despite this, the rate of iron uptake increased by only 20% in the transformed cells due to less efficient cycling of the transferrin receptors and less efficient release of iron from transferrin to intracellular sites. Some internalized iron was released from the transformed cells still bound to transferrin. A fast and a slow rate of transferrin exocytosis were identified in transformed cells, as in normal cells, indicating that there were at least two intracellular pathways for transferrin. The fast pathway predominated in the transformed cells, compared with an equal importance of the two pathways in the normal cells.

Animals↗

PubMed1989

A method for infection of cultured myogenic cells with Rous sarcoma virus using polybrene.

A method for efficiently infecting primary myogenic cultures with a temperature-sensitive variant of the Prague strain of Rous sarcoma virus (RSV, tsLA24) to obtain a high yield of transformed myogenic cells is reported. It incorporates the use of an amorphous polymer of polycations, Polybrene, to enhance the absorption of the virus by the muscle cells. In addition, other steps which were shown to be important were a) to allow cell attachment before infection, b) to infect at 35 degrees C in low protein medium, c) to use a density of 1 to 1.5 X 10(6) cells/60-mm dish, d) gentle agitation during infection, and e) to minimize the number of passages after infection. The use of the temperature-sensitive virus provided a means of confirming the presence of myogenic cells in transformed cultures. When infected cells were maintained at 35 degrees C (the permissive temperature for virus activity) they exhibited the characteristics of transformed cells. These characteristics included altered cell morphology, the absence of contact-inhibited growth, growth in semisolid medium, and expression of the src oncogene. In contrast, when infected cells were maintained at 41 degrees C (the nonpermissive temperature for virus activity) they do not express src and showed normal myogenic development and ultimately formed myotubes.

Animals↗

PubMed1988

Differences in transferrin receptor function between normal developing and transformed myogenic cells as revealed by differential effects of phorbol ester on receptor distribution and rates of iron uptake.

The effects of the tumor promotor, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), on the intra- and extracellular distribution of transferrin receptors and rates of iron uptake were studied in normal developing myogenic cells and myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus. In normal developing cells PMA was found to increase the rate of iron uptake by 15-30%. There was, however, no effect on transferrin receptor distribution, suggesting that the increase in iron uptake was due to stimulation of the rate of receptor cycling. In contrast, in transformed myogenic cells, PMA had no effect even at concentrations 10 times those effective in normal myogenic cells. The specificity of PMA was demonstrated by comparison with 4 alpha-phorbol which had no effect compared with the control cells which were incubated with dimethyl sulfoxide, the solvent used to dissolve the phorbols. These results indicate a functional difference in the transferrin receptor between normal and transformed myogenic cells. The data for normal myogenic cells are similar to those previously reported for normal erythroid cells, but differ from those for some transformed cell lines in which phorbol esters were shown to cause internalization of transferrin receptors.

Animals↗

PubMed1988

Transferrin endocytosis and iron uptake in developing myogenic cells in culture: effects of microtubular and metabolic inhibitors, sulphydryl reagents and lysosomotrophic agents.

The experiments described in this study were designed to investigate receptor-mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts (dividing and non-dividing) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor-mediated endocytosis of the protein.

2,4-Dinitrophenol↗

PubMed1988

Species specificity of transferrin binding, endocytosis and iron internalization by cultured chick myogenic cells.

The ability of unlabelled heterologous transferrin to interact with transferrin receptors on developing chick myogenic cells was investigated by measuring their capacity to inhibit the surface-binding and internalization of 125I- and 59Fe-labelled ovotransferrin. Transferrins from rat, rabbit, human, and a species of kangaroo (Macropus fuliginosus) were unable to inhibit either surface-binding or internalization of labelled ovotransferrin even at concentrations ten times the molar concentration of the ovotransferrin. Transferrins isolated from the serum of a toad (Bufo marinus) and a lizard (Teliqua rugosa), when added at high concentrations, were found to reduce surface-binding of 125I-Tf by 20-25% but did not inhibit internalization of either 125I-Tf or 59Fe. This suggests that the effects of toad and lizard transferrins are due to non-specific binding to the myogenic cells. In contrast, inhibition of both surface-binding and internalization of labelled ovotransferrin was found when myogenic cells were incubated in the presence of the homologous transferrin (ovotransferrin). The species-specificity of transferrin binding, endocytosis and iron internalization did not vary with the state of proliferation or differentiation of the myogenic cells. However, the intracellular iron utilization was found to differ between differentiating presumptive and terminally differentiated myotubes. Internalized 59Fe was fractioned by gel filtration. In dividing and non-dividing presumptive myoblasts 59Fe was found to elute in three peaks, two with elution volumes corresponding to ferritin and transferrin and one at greater elution volume than that of myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development.

The mechanism of iron uptake and the changes which occur during cellular development of muscle cells were investigated using primary cultures of chick embryo breast muscle. Replicating presumptive myoblasts were examined in exponential growth and after growth had plateaued. These were compared to the terminally differentiated cell type, the myotube. All cells, regardless of the state of growth or differentiation, had specific receptors for transferrin. Presumptive myoblasts in exponential growth had more transferrin receptors (3.78 +/- 0.24 X 10(10) receptors/micrograms DNA) than when division had ceased (1.70 +/- 0.14 X 10(10) receptors/micrograms DNA), while myotubes had 3.80 +/- 0.26 X 10(10) receptors/micrograms DNA. Iron uptake occurred by receptor-mediated endocytosis of transferrin. While iron was accumulated by the cells, apotransferrin was released in an undegraded form. There was a close correlation between the molar rates of endocytosis of transferrin and iron. Maximum rates of iron uptake were significantly higher in myotubes than in presumptive myoblasts in either exponential growth or after growth had plateaued. There were two rates of exocytosis of transferrin, implying the existence of two intracellular pathways for transferrin. These experiments demonstrate that iron uptake by muscle cells in culture occurs by receptor-mediated endocytosis of transferrin and that transferrin receptor numbers and the kinetics of transferrin and iron uptake vary with development of the cells.

Animals↗