Although antibodies against nuclear antigens are considered the hallmark of systemic lupus erythematosus (SLE) 1, antibodies against cytoplasmic constituents have also been identified (1, 2). Despite their recognition more than 25 years ago, the prevalence, antigenic specificities, and disease associations of antiribosomal antibodies (ARA) are all controversial (3-10). Since these problems may relate to detection of different ribosomal or ribosome-associated antigens, we attempted to define more precisely the identity of the ribosomal protein antigens. The results of this study indicate that ARA reactive with ribosomal proteins occur in ~5-10% of SLE patients, but are highly specific, binding to epitopes on 3 out of a total of-80 proteins. Materials and MethodsAntibody Screening Procedures. Cellular localization of antigens was determined by indirect immunofluorescence using Hep-2 cells as substrate. The effect of the enzymes RNase (50 #g/ml), trypsin (5 ~zg/ml), and proteinase K (0.5 ~g/ml) on the intensity of immunofluorescence was quantitatively analyzed using a FIAX fluorometer, kindly loaned by Dr. L. J. Kagen (The Hospital for Special Surgery). Reactivity with saline-soluble extracts of human spleen and rabbit thymus was determined by counterimmunoelectrophoresis (CIE) (11) using standard reference serum to Ro, La, Sm, and ribonucleoprotein (RNP) (12). An anti-Jo-1 reference serum was kindly provided by C. C. Bunn and G. R. V. Hughes, Hammersmith Hospital, London, Great Britain.Isolation oJ Ribosomes. Ribosomes were isolated from dog, rat, and chicken livers essentially by the method of Fairhurst et al. (13). Briefly, livers were homogenized in 0.25 M sucrose in TK2~M buffer (20 mM Tris HCI, 25 mM KCI, 5 mM MgCI2, pH 7.5). The postmitochondrial supernatant was adjusted to 1.0% deoxycholate and centrifuged through a discontinuous sucrose gradient using an SW40 rotor. In some cases, the KC1 concentration in the sucrose was increased to 500 mM. The ribosomal pellet was washed and resuspended in distilled water or an appropriate buffer.Analysis of Ribosomal Constituents. The isolated ribosomes had an optical density 260/280 ratio of ~ 1.6. The RNA composition was analyzed by sucrose density ultracentrifugation on a 5-20% linear gradient using a SW60 rotor, as well as by a 2% composite gel (14) in a vertical gel apparatus (15). The ribosome was separated into large and small subunits either by incubation in 20 mM EDTA for 10 rain at room temperature, or by This work was supported by grants AM 32845 and AM 14627 from the National Institutes of Health, Bethesda, MD, and by grants from the Lupus Foundation of America. Address correspondence to Keith B. EIkon, The Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021.i Abbreviations used in this paper: ARA, antiribosomal antibodies; CIE, counterimmunoelectrophoresis; CM-cellulose, carboxymethylcellulose; NEPHGE, nonequilibrium pH gel electrophoresis; RNP, ribonucleoprotein; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLE, sys...

Tamm-Horsfall (T-H) glycoprotein was demonstrated in the developing hamster kidney using immunofluorescence and immunoelectron microscopical techniques. The glycoprotein was first observed in the fetal kidneys on the 12th day of gestation and was confined to the luminal surface of the presumed distal tubules of the medulla. It was not until the 14th day of gestation that T-H glycoprotein was also sometimes seen to be associated with the lateral and basal invaginations of the plasma membranes of the now differentiated distal tubules. On the 16th day (1st day post-partum) the glycoprotein was also found in the cortex. Although the general distribution of T-H gylocoprotein was at 3–4 days after birth similar to the adult, the full intensity of staining was not attained until after the 21st day. The possible physiological significance of these findings is discussed.

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.

Greatly enhanced secretory activity was observed during pregnancy and during lactation in the adenohypophysis ofthe rabbit, in cells similar to those designated type 1 in normal animals. These cells, whose granules were up to 550 m\g=m\ in diameter, showed, under the electron microscope, a very well developed ergastoplasm, an enlarged Golgi region, frequent nuclear inclusions and, during lactation, extensive degranulation. These observations, taken together with the evidence provided by light microscopy (Allanson, , suggest that the type-1 cells are the producers of prolactin.