George S. Eisenbarth

Hormonal Regulation of Cartilage Growth and Metabolism

Publisher Summary This chapter describes the hormonal regulation of cartilage growth and metabolism. The central role of cartilage in linear growth has been appreciated as the process of endochondral ossification was recognized. Cartilage is a specialized connective tissue whose primary function is to make a matrix that is resilient and resists deformation and compression. Several different types of cartilage exist in animals, and each appears to be adapted to its own specific function by the chemical composition of its matrix. The matrix compositions of the three major types of cartilage, elastic, fibrous, and hyaline are studied. Most of the studies of the effect of serum sulfation factor on cartilage metabolism in vitro have been conducted using serum containing sulfation factor activity or a partially purified fraction of serum containing such activity. It is shown that serum sulfation factor probably stimulates cartilage amino acid transport through a CAMP-dependent mechanism. It is demonstrated that serum sulfation factor increases cartilage cAMP content as well as α-aminoisobutyrate transport and that the correlation between the two effects is striking.

Prostaglandin A1 inhibition of chondrosarcoma growth

The effects of various prostaglandins (PG) on the in vitro synthesis of macromolecules by two transplantable chondrosarcomas were studied. Prostaglandin A1 markedly inhibited the incorporation of radioactive precursors into chondromucoprotein, total protein, RNA and DNA of both a well differentiated rat chondrosarcoma and a poorly differentiated murine chondrosarcoma. PGE1 and PGF1α had no effects on the synthesis of macromolecules by either tumor. PGA1 inhibitory effects occurred over a dose range of 1 to 25 μg/ml. PGA1 had no effect on the synthesis of macromolecules by liver. The data indicate that malignant transformation of cartilage cells does not alter their responsiveness to PGA1.

Production of Monoclonal Antibodies Reacting with Rat Islet Cell Membrane Antigens

The present communication describes the generation of twelve lymphocyte hybrid cell lines whose antibodies react with the rat islet cell line RIN, and the initial characterization of the tissue specificity and functional properties of these antibodies. Since antibodies recognizing islet cell differentiation antigens were sought, these permanent hybrid cell lines were developed from cultures whose antibodies, by radioimmunoassay, bound minimally or not at all to rat red blood cells (cell line A3C1 is an exception). Antibodies from five of the cell lines by radioimmunoassay react significantly with cultured rat fibroblasts in addition to their reaction with RIN cells. Antibody F41-5D6 by indirect immunofluorescence reacted with sections of the RISL transplantable islet cell tumor and specifically with islet cells in sections of rat pancreas. Four antibodies (F41-1G3, 5B5, 6C5, and 5A5), by indirect immunofluorescence, reacted with sections of the RISL transplantable islet cell tumor but not with sections of normal pancreas. Seven of the antibodies were cytotoxic to cultured RIN cells and seven antibodies share the useful property of reacting with protein A. This study demonstrates the feasibility of producing monoclonal antibodies to islet cell differentiation antigens and describes several antibodies which should be useful reagents in studies of the physiology and pathophysiology of the islet cell plasma membrane.

[30] Production of monoclonal antibodies reacting with the cytoplasm and surface of differentiated cells

Publisher Summary This chapter describes the techniques that have been successful in creating monoclonal antibodies to neuronal T cell, islet cell and thymic cell antigens. The techniques involve a minimum of tissue culture work, as it finds it unnecessary to feed the cultures (except for the addition of a single drop of medium at 7 days) until colonies are visible and ready for assay. A major advantage of the monoclonal antibody system is the ability to immunize with only partially purified antigens (e.g., whole cells). There are several cross-reactivities of existing monoclonal antibodies with unexpected tissues (e.g., anti-T cell antibody 4F2 reacting with pancreatic islet cells). Thus, a monoclonal antibody may already exist that will serve the function (e.g., cell isolation) for which an investigator is considering monoclonal antibody production.

Fatty acid inhibition of somatomedin (serum sulfation factor)-stimulated protein and RNA synthesis in embryonic chicken cartilage

Abstract The effects of fatty acids and serum containing somatomedin on in vitro synthesis of collagen, chondromucoprotein, general proteins and RNA were studied in embryonic chicken cartilage. Somatomedin (5 % rat serum added) stimulated 35 SO 2− 4 , [ 3 H]leucine and [ 3 H]tryptophan incorporation into chondromucoprotein-rich and collagen-rich cartilage protein fractions; and [ 3 H]proline incorporation and [ 3 H]hydroxyproline synthesis in the collagen-rich fraction. Somatomedin also stimulated [ 3 H]uridine incorporation into RNA. In the non-stimulated state (no serum added) butyrate and octanoate had little or no effect on amino acid incorporation into protein or uridine incorporation into RNA. In the serum stimulated state, butyrate and octanoate inhibited amino acid incorporation into protein and uridine incorporation into RNA. Such inhibition was observed only with serum containing growth hormone-dependent sulfation factor (somatomedin). Agarose column chromatography of the chondromucoprotein-rich fraction revealed that while somatomedin stimulates synthesis of all proteins of this fraction equally, butyrate inhibits stimulated chondromucoprotein synthesis to a greater extent than certain other cartilage proteins (which are inhibited little, if at all). The data indicate that somatomedin stimulates embryonic chicken cartilage RNA, collagen, chondromucoprotein and general protein synthesis. Fatty acids have little or no effect on unstimulated cartilage macromolecular synthesis. The somatomedin stimulation of RNA, collagen and chondromucoprotein synthesis, and some, but not all of protein synthesis other than collagen or chondromucoprotein, is inhibited by fatty acids.

Prostaglandin inhibition of cartilage chondromucoprotein synthesis: Concept of “intrinsic activity”*

We have recently reported that cartilage has two sites for prostaglandin (PG) action. One site (S1) is stimulated by PGA1, PGE1 and PGF1α and elevates tissue cyclic 3′5′adenosine monophosphate (cAMP). A second site (S2) is activated by PGA1 (but not PGE1 or PGF1α) and inhibits the synthesis of cartilage macromolecules. The present study is an investigation of the effects of PGB1 on embryonic chicken cartilage chondromucoprotein synthesis in vitro. PGB1 was found to inhibit chondromucoprotein synthesis with an apparent affinity for S2 which was similar to that of PGA1. The maximal inhibition produced by PGB1 was, however, approximately one-half the maximal inhibition caused by PGA1. Studies of the combined effects of PGB1 and PGA1 were consistent with the hypothesis that both classes of prostaglandins act at a common site (S2) with about equal affinity but that PGB1 has a lower intrinsic activity than PGA1. Similar studies of the combined effects of PGE1 or PGF1α with PGA1 indicate that neither PGE1 nor PGF1α binds significantly to S2. An independent effect of PGB1 to activate S1 and elevate tissue cAMP was also found.

Evidence that endogenous cyclic AMP does not modulate serum sulfation factor action on embryonic chicken cartilage.

The role of cartilage cyclic AMP as a mediator or modulator of serum sulfation factor (SSF) action on embryonic chicken cartilage was assessed. Media with concentrations of rat serum (7.5%) sufficient to maximally stimulate chondromucoprotein synthesis as measured by 35SO4 incorporation did not change cartilage cyclic AMP levels. Theophylline (2.5mM) doubled cyclic AMP in cartilage incubated in media but had no effect on 35SO4 incorporation. In media containing 5% rat serum, theophylline at 0.5, 1.5 and 2.5mM caused a similar and significant rise in tissue cyclic AMP but only 2.5mM inhibited SSF stimulated 35SO4 incorporation. The data indicate that cartilage cyclic AMP neither mediates nor modulates SSF action on cartilage chondromucoprotein synthesis.

Polyethylene Glycol in Suppositories: Carcinogenic?

Excerpt To the editor: Polyethylene glycol, a family of chemical compounds composed of linear polymers of ethylene oxide and water, is a component in many medications, including ointments, where it...

Lymphocyte suppressor activity in patients with polyglandular failure

The Con-A--activated suppressor function of lymphocytes from polyglandular failure (PGF) patients in human mixed lymphocyte culture was compared to a normal population. As a group, PGF patients were found to have decreased suppressor activity: 67% of normal for autologous suppression, 45% of normal for heterologous. However, lymphocytes from most PGF patients have neither an absolute lack of suppressor activity nor an absolute inability to respond to suppression. The marked variability of assayed suppression, depending on the combination of stimulators and responder cells tested, limit the utility of this assay in defining individuals with abnormal suppressor function. One patients lymphocytes were unique in that although suppression of heterologous cells was normal, suppression of her own cells was defective. Defective suppressor function may be related to the susceptibility of these patients to multiple autoimmune diseases.

A visual assay to monitor purification of cell surface antigens reacting with monoclonal antibodies

We have developed a visual microtiter assay to detect solubilized cell-surface antigens which react with monoclonal antibodies. The assay depends on the ability of adsorbed monoclonal antibody to bind target cells to microtiter V wells, and the inhibition of binding by antigen. We have used this assay to follow a 600-fold purification of the human lymphocyte differentiation antigen 3A1 extracted from HSB-2 cells. Antigen 3A1 is a glycoprotein with a molecular weight of approximately 40,000 daltons.