Robert W. Chesnut

Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma: Relationship of the major sialoglycoprotein to xenotransplantability

Abstract The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-C1) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [ 3 H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also labeled by lactoperoxidase and 125 I, indicating its presence at the cell surface. Metabolic labeling with [ 3 H]glucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantable (MAT-C1) and non-xenotransplantable (MAT-B1) sublines, representing >70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labeling and transmission electron microscopy (TEM) do not show substantially greater amounts of negatively charged groups distributed along the cell surfaces of MAT-C1 than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.

Water in normal muscle and muscle with a tumor.

The total water content, the amount of non-freezable water, and the Na-+ and K-+ contents in the gastrocnemius muscle of albino mice with and without a solid tumor were determined. The spin-lattice relaxation time (T1) for the water protons in the two kinds of muscle were measured at six resonance frequencies ranging from 4.5 to 60 MHz over the temperature range +37 to minus 65 degrees C. Quantitatively calculated T1 values are given. The difference in T1 for the two types of muscle at temperatures above minus 5 degrees C is attributed to the difference in the distribution ratio of water between hydration and free states, and bears no direct relation to the concentration of Na-+.

Antibacterial activity of 15-azasteroids alone and in combination with antibiotics.

A new class of 15-azasteroid analogues has been synthesized and tested for antimicrobial activity. The compounds 1, 10, 11, 11a-tetrahydro-7-methoxy-11a-methyl-2H-naphth (1,2-g) indol (methoxyimine) and 1,10,11,11a-tetrahydro-11a-methyl-2H-naphth (1,2-g) indol-7-ol (hydroxyimine) inhibit the growth of Bacillus subtillis and Escherichia coli at concentrations as low as 10-5 M. Addition of either compound to the growth medium casued a rapid inhibition in the transport of radioactive glucose, uracil and several amino acids. The inhibition of growth and substrate transport was reversed following removl of the steroid from the medium. The evidence is consistent with a site of steroid action at the cell periphery. Combining the methoxyimine with polyor circulin at subinhibitory concentrations produced greatly enhanced antimicrobial activity against Pseudomonas fluorescens. Similar action was observed against B. subtilis when the azasteroid was combined with vancomycin or chloramphenicol. The inhibitory action of other antibiotics such as penicillin or erythromycin was not affected by addition of the test compound. The results suggest formation of a molecular complex between the azasteroid and antibiotic which is responsible for the enhanced biological activity.

Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells.

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min. and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8-20 h period. Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstanital evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.

Triaryl-substituted Schiff bases are high-affinity subtype-selective ligands for the estrogen receptor.

We have explored the isoelectronic replacement of the C=C double bond found at the core of many nonsteroidal estrogen ligands with a simple Schiff base (C=N). Di- and triaryl-substituted imine derivatives were conveniently prepared by the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these imines profiled as ERα agonists but as ERβ antagonists, showing preferential reliance on the N-terminal activation function (AF1), which is more active in ERα. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way: by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional coactivators. This work suggests that C=N for C=C substitution might be more widely considered as a general strategy for preparing drug analogues.

Purification of plasma membranes of rat mammary gland: Comparisons of subfractions with rat milk fat globule membrane☆

Abstract Partially purified plasma membranes of rat mammary gland, obtained as light (F 1 ) and heavy (F 2 ) fractions by flotation on a discontinuous sucrose density gradient, were further fractionated by density perturbation flotation using digitonin to shift the density of the cholesterol-rich portion of the membranes. The shifted fraction (F 1 F 3 ) of digitonin-treated F 1 was highly enriched in 5′-nucleotidase, cholesterol and sialic acid, but free of galactosyltransferase, suggesting that it contained highly purified plasma membranes. The unshifted fraction (F 1 DF 1 ) was enriched in galactosyltransferase and depleted in nucleotidase, cholesterol and sialic acid, suggesting that it contained Golgi fragments. The F 2 fraction shows substantially different behavior. Part of it re-equilibrates to the F 1 position upon reflotation. When treated with digitonin, part of F 2 is shifted to a higher density (F 2 DF 3 ). F 2 DF 3 is enriched in 5′-nucleotidase, cholesterol, sialic acid and galactosyltransferase. These properties suggest that this subfraction comes from a plasma membrane containing galactosyltransferase. The sialoglycoproteins of the various fractions were compared with those of rat milk fat globule membrane, which is derived in part from the apical surface of the mammary secretory cell. Dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals two major glycoprotein bands (GP-II and GP-III) in F 1 DF 3 . F 2 DF 3 contains these and an additional band of lower mobility (GP-I). Both crude and purified MFGM contain all three bands. Comparisons of peanut lectin receptors by autoradiography of polyacrylamide gels run in SDS and then treated with [ 125 I]peanut lectin also suggest that F 2 DF 3 is more similar to the milk fat globule membrane than is F 1 DF 3 . However, analysis of the membrane polypeptides and concanavalin A (ConA) receptors shows no obvious relationship between milk fat globule membrane and any of the isolated mammary membrane fractions. These results indicate that the relationship between the milk fat globule membrane and mammary membranes is complex, possibly involving components not associated with the mammary plasma membrane or only selected components of the plasma membrane.

Changes in expression of a major sialoglycoprotein associated with ascites forms of a mammary adenocarcinoma

Glycoproteins of a cultured form (MR) of the 13762 rat mammary adenocarcinoma and its variants have been studied by analyses for peanut agglutinin receptors, [3H]glucosamine labeling, lactoperoxidase labeling and CsCl density gradient centrifugation. The 13762 MR cells, derived from 13762 MAT-B ascites cells, do not contain detectable ASGP-1, the predominant cell surface sialoglycoprotein of the ascites forms of the 13762 tumor. Transplantation and continued passage as ascites cells of MR cells or clonal lines derived from MR results in abrupt expression of ASGP-1 at about passage 16; it is absent in early passages of the ascites tumor. When these ascites cells are transferred to culture, ASGP-1 is again lost. No ASGP-1 is found in solid tumors derived from subcutaneous transplantation of the 13762 MR cells. The results suggest modulation of ASGP-1 content of the 13762 tumor cells.

Effect of 15-azasteroid analogues on cell culture growth

Two azasteroid analogues, 1,10,11,11a-tetrahydro-11a-methyl-2H-naphth [1,2-g] indol-7-ol (compound I), and 1,10,11, 11a-tetrahydro-7-methoxy-11a-methyl-2 H-naphth[1,2-g] indole (compound II) inhibit the growth of KB and L-M cells in culture. There is a structural specificity for biological effectiveness with compound I being more inhibitory at equivalent concentrations. When compound I is present during growth of KB and L-M cells, the permeability of the lysosomal membrane is increased. Compound I reduced in a nearly equivalent manner the transport of precursors into the cells for all three macromolecular species, protein, DNA and RNA. The action of compound I can be accounted for by an action upon the cell membrane.

15-Azasteroid blockage of cell permeability and mitochondrial respiration

The 15-azasteroid, 1,10,11,11a-tetrahydro-11a-methyl-2H naphth (1,2-g)indol-7-o1, inhibits the growth of the cell culture lines KB and L-M as well as several strains of bacteria. The inhibition of growth is reversed following removal of the steroid from the growth medium. Using in vitro grown L-M cells, the compound inhibited the transport of amino acids and uracil. The action was non-detergent like and at least 100 times more effective in terminating metabolite transport than sodium azide. The azasteroid inhibited the oxidation of glutamate in isolated rat liver mitochondria. The oxidation of succinate was not effected by the azasteroid alone but in the presence of glutamate, the azasteroid uncoupled the oxidation of succinate from the ADP-ATP control. It is suggested that the azasteroid may be acting directly on the electron transport system and/or acting indirectly through membrane perturbations which disrupts the electron transport process.

Glycoprotein differences between the ascites and cultured forms of the sarcoma 180 tumor

Summary The cell surface glycoproteins of Sarcoma 180 tumor cells grown in vitro in cell culture or in vivo as an ascites form in mice have been examined by periodate-Schiff staining of dodecyl sulfate acrylamide gels and by lactoperoxidase labeling. The cells grown in culture show the presence of a set of surface membrane glycoproteins which are absent or markedly decreased in the ascites form. Conversely, the ascites form has a glycoprotein which is not evident in the cells in culture. The other surface membrane polypeptides and enzyme activities are essentially the same for the two cell types. The glycoprotein differences may be related to the ability of the cells to interact with each other or a substratum.