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Biomedical subjects

S Ratner

Publications and source records attributed to S Ratner.

At least 37 records · Page 2Linked to original sources
PubMed1988

Motility and tumoricidal activity of interleukin-2-stimulated lymphocytes.

The motility of murine splenic lymphocytes stimulated nonspecifically by recombinant interleukin 2 (RIL-2) was studied in a three-dimensional collagen-gel system. Nonadherent BALB/c splenic lymphocytes were cultured in medium containing Cetus RIL-2 (700 to 1000 units/ml) or excipient control. They were then allowed to locomote randomly for 16 to 18 h into slabs of type I rat tail collagen gel. The gels were digested with collagenase, and total lymphocyte populations and motile subpopulations were collected and compared with respect to their lymphokine-activated killer activity (measured as 4-h cytotoxicity against the natural killer-resistant mammary adenocarcinoma line 410.4), their natural killer activity (measured as 4-h cytotoxicity versus lymphoma YAC-1), and their subset distribution (defined by immunofluorescence). Some of the slabs were not digested but fixed for measurement of leading-front distance. RIL-2-stimulated lymphocyte populations displayed greater motility than unstimulated populations; the mean leading front distance was 2.4 times greater, and the percentage of cells exhibiting motility was approximately doubled. The most motile RIL-2-stimulated cells, however, were not the most tumoricidal. Motile subpopulations displayed approximately 25 to 60% lower lymphokine-activated killer activity than did the total populations from which they were derived. Natural killer activity followed a similar pattern. Motile subpopulations contained a lower proportion of asialo-GM1+ and T-null cells than did total populations and a higher proportion of L3T4+ cells. Chemokinetic stimulation with alpha-interferon increased overall motility, but the lymphokine-activated killer activity of the motile subpopulation was still lower than that of the total population. Lymphocyte motility is important in the infiltration of tumors and other inflammatory lesions. The results indicate that the most tumoricidal lymphocytes in RIL-2-stimulated populations may not be the best tumor infiltrators, and that the tumoricidal activity of circulating lymphocytes may be a misleading indicator of the effectiveness of immunotherapy.

Animals↗
PubMed1988

Motility of murine lymphocytes during transit through cell cycle. Analysis by a new in vitro assay.

The relationship between the basal (spontaneous) motility of murine lymphocytes and their position in the cell cycle was examined in a new collagen gel motility assay system. Concanavalin A-stimulated or control lymphocytes were allowed to locomote into slabs of type I collagen gel. The assay configuration permitted extraction of both total populations and locomotory subpopulations as viable, single-cell suspensions suitable for phenotypic and cell analysis. Concanavalin A stimulation resulted in a significant increase in the mean distance traveled by the leading cell front in 4 hr, from 23 microns (controls) to 67 microns. The estimated percentage of motile cells increased from 0.9 to 2.8%. Similar increases were observed after 18 hr of locomotion. The SIg+, Thy-1+, L3T4+, and Ly-2+ subsets exhibited equivalent increases in motility. Total populations and locomotory subpopulations were allowed to incorporate 5-bromo-2'-deoxyuridine, and their cell cycle profiles were compared by dual parameter anti-5-bromo-2'-deoxyuridine, propidium iodide fluorescence analysis. Total population and locomotory subpopulations did not differ significantly with respect to the ratio G0/G1:S, indicating that lymphocytes in these two phases exhibited approximately equal motility. Cells in late S and G2 + M were significantly less motile; locomotory subpopulations contained 60 to 75% fewer G2 + M cells than the total populations from which they were derived. Taken together, the results indicate that the concanavalin A-induced increase in motility commences before S phase and that motility diminishes shortly before or during G2 + M.

Animals↗
PubMed1987

A new assay to measure monoclonal antibody-dependent complement or macrophage-mediated tumor growth inhibition.

A new in vitro assay has been developed to measure the inhibition of tumor growth by antibody and complement or by antibody and macrophages. Tumor cells (1 X 10(5) cells) or a mixture of tumor cells (1.5 X 10(4) cells) and macrophages (1.5 X 10(5) cells) are immobilized in a 1 microliter agarose droplet. Antibody is added to the medium bathing the agarose droplet. Complement is also added to wells containing droplets with tumor cells alone. The area covered by tumor cell monolayer is measured non-destructively from day 0 to day 7 with a split image tracing device. Cell growth is expressed by the increase in the square root of the measured area. This assay does not require isotopes and can be used to test tumor cells freshly dissociated from solid tissues. It permits cell-cell interactions which may change the sensitivity of tumor cells to various treatments. The extended period of observation allows the testing of multiple treatments. Surviving cells can also be recovered for further study. This assay may be useful for testing the efficacy of monoclonal antibody treatment on solid tumors.

Animals↗
PubMed1987

Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site.

We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase, EC 4.3.2.1) with the active site-directed reagent bromo[U-14C]mesaconic acid, an analogue of fumaric acid. Reactivity, measured by enzyme inactivation, followed pseudo-first-order kinetics, and the rate increased with reagent concentration. Argininosuccinate completely protected the enzyme against inactivation, but neither arginine nor fumarate was protective. A plot of the degree of inactivation as a function of alkyl groups incorporated was extrapolated to 4 mol per mol of enzyme, or 1 mol per active site. After large-scale alkylation of the enzyme (and digestion with trypsin), two 14C-labeled tryptic peptides were isolated. These were chemically sequenced by the Edman method. The amino acid sequences proved to be identical with regions of the deduced amino acid sequences or argininosuccinases from human and yeast sources [O'Brien, W. E., McInnes, R., Kalumuck, K. & Adcock, M. (1986) Proc. Natl. Acad. Sci. USA 83, 7211-7215; Beacham, I. R., Schweitzer, B. W., Warrick, H. M. & Carbon, J. (1984) Gene 29, 271-279]. The 14C-labeled tryptic peptide in the active site region had the sequence Gly-Leu-Glu-Xaa-Ala-Gly-Leu-Leu-Thr-Lys; Xaa represents an unknown phenylthiohydantoin derivative detected in cycle 4. The corresponding amino acid was identified as lysine-51 on the basis of sequence similarity with human and yeast amino acid sequences in this region. The reaction of the enzyme with the alkylating agent and the specific protection against inactivation by argininosuccinate suggest that this lysine residue has an essential role in the binding of argininosuccinate to the enzyme and, consequently, is essential for catalysis.

Amino Acid Sequence↗
PubMed1986

Inflammatory infiltrates of experimental mammary cancers.

The purpose of this review was to summarize observations on the type and function of inflammatory infiltrates of mouse mammary tumors and to speculate on the underlying mechanisms and the significance of infiltrates to mammary tumor biology. Although the major conclusion is that much more work is needed, certain themes seem to be emerging. The number of infiltrating cells can be very high but is unrelated to biological behavior of the tumors. What seems to be important is the relative contributions of inflammatory cell subsets. In the case of T-cell subsets and NK cells, the infiltrates from tumors of long-term cell lines so far seem uninformative. The general characteristics are similar to those of infiltrates from rapidly proliferating, normal mammary tissues. These characteristics do not correlate with diverse biological behavior or malignant potential. A more informative model appears to be one in which the development of tumors from preneoplastic tissue can be observed. Here our attention is currently focused on NK cells. By contrast, the correlation between activated TAM and metastatic behavior suggests that our transplantable MMT lines may be biologically relevant in the study of infiltrating macrophages. We are especially interested in the role of TAM in the generation of tumor cell variability. Overall, our data indicate that the host infiltrate is another manifestation of both inter- and intra-tumor heterogeneity and, as such, is not simply a response to, but, rather, a part of the tumor ecosystem. Unraveling the cellular and molecular mechanisms that govern the inflammatory cell component of tumors should provide insight into the types of cellular interactions that result in tumor development and progression.

Animals↗
PubMed1986

Analogous recognition of phospholipids by insect phagocytes and mammalian macrophages.

Phagocytic cells from larvae of the moth Heliothis virescens and peritoneal macrophages of mice were observed to preferentially bind negatively charged phospholipid vesicles containing phosphatidylglycerol or phosphatidylserine as compared to neutral or positively charged vesicles. Since phagocytes have retained their primitive function of endocytosis throughout evolution, the recognition of negatively charged phospholipids may be a primitive mechanism for the identification of potential targets by macrophages.

Animals↗
PubMed1985

Argininosuccinate synthetase: essential role of cysteine and arginine residues in relation to structure and mechanism of ATP activation.

We have undertaken studies to identify amino acid residues that are involved in the catalytic mechanism of argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] and have found that a cysteine residue and an arginine residue are required for activity. The reactive cysteine residues are accessible to solvent and available to react with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Four cysteine residues, one per subunit, are shown by enzymatic assay to be required for catalytic activity, suggesting that a reactive cysteine lies within the active site of argininosuccinate synthetase. In the presence of sodium dodecyl sulfate, 12 cysteine residues react with DTNB; consequently, all of the half-cystine residues in the native enzyme are present in the reduced sulfhydryl form. We also present evidence for the participation of arginine groups in the binding of ATP and PPi. Modification of argininosuccinate synthetase with [14C]-phenylglyoxal results in incorporation concomitant with loss of catalytic activity of 4 mol of phenylglyoxal per mol of native enzyme (one arginine per active site). ATP and PPi protect the enzyme from phenylglyoxal incorporation. Based on these results, we propose that the essential arginine in the active site participates in the binding of ATP and PPi. The binding of ATP and PPi at the same site is mutually exclusive; this exclusion is in accord with the finding that argininosuccinate synthetase has one reactive arginine residue per active site per subunit. This is consistent with our previously proposed reaction mechanism.

Adenosine Triphosphate↗
PubMed1985

T cell locomotion in the tumor microenvironment. I. A collagen-matrix assay.

Previous studies have shown that lymphocytic infiltrates of different mouse mammary tumors contain different proportions of the T cell subsets Lyt-1+ and Lyt-2+. These characteristic subset ratios may be established, at least in part, by differential locomotion of subsets in response to components of the tumor microenvironment, such as soluble chemotactic and chemokinetic factors, cell and stromal surfaces, oxygen tension, and pH. We describe a new in vitro assay for determining how such microenvironmental variables affect lymphocyte locomotion. Suspended lymph node cells, alone or mixed with other cell types, are sandwiched between two layers of type I collagen gel and bathed in culture medium. A halo of locomotory cells fans out around the flattened droplet. Locomotion requires energy and exogenous protein. After a 24-hr incubation at 37 degrees C, 10% CO2 in air, the cell density of the halo is analyzed optically and the subset ratios are characterized by immunofluorescence staining of the gel sandwich. Under standard culture conditions, the locomotory population is enriched 12% in Thy-1+ cells compared with the bulk population, and the ratio of Lyt-1+ to Lyt-2+ cells is significantly increased. Lymphocyte locomotion is inhibited by 1 microM PGE2, by decreased pH and oxygen tension, and by the presence of normal mammary cells or mammary adenocarcinoma cells. The Lyt-1+:2+ ratio in the locomotory population is not altered by PGE2 but is reduced by acidity and hypoxia. The ratio is also reduced by the presence of mammary cells and cells of one of the mammary adenocarcinoma cell lines tested (168) but not by two others (68H and 410). Our data support the hypothesis that the locomotion of T cell subsets is differentially responsive to the types of microenvironmental conditions that vary among tumors.

Animals↗
PubMed1983

A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate.

A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions.

Animals↗
PubMed1982

Argininosuccinate synthetase of bovine liver: chemical and physical properties.

Homogeneous crystalline argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] prepared from bovine liver according to Rochovansky et al. [Rochovansky, O., Kodowaki, H. & Ratner, S. (1977)J. Biol. Chem. 252, 5287-5294] has been characterized with respect to amino acid composition and other chemical and physical properties. The total residue molecular weights derived from the amino acid analysis are in agreement with values previously obtained by physical means for the catalytically active tetramer, 185,000, and the monomer, 46,500. The enzyme is focused sharply at pH 7.6 as a single protein. Additional properties reported include 2.74 X 10(5) M-1 cm-1 for the molar absorption coefficient, based on the absolute value for protein, and 0.747 ml/g for the chemically based partial specific volume.

Amino Acids↗