In 18 of 20 patients with psychosis secondary to systemic lupus erythematosus (SLE), autoantibodies to ribosomal P proteins were detected by immunoblotting and measured with a new radioimmunoassay using a synthetic peptide as antigen. The frequency of anti-P was not increased in patients with other central nervous system manifestations of SLE (3 of 20, by radioimmunoassay), in patients with transient behavioral abnormalities due to SLE (none of 8), in patients with psychosis who did not have SLE (none of 13), or in normal controls (none of 20). In four of five paired serum samples, anti-P-peptide antibody levels increased 5-fold to 30-fold during the active phase of lupus psychosis. Longitudinal studies of anti-P activity in two patients with psychosis revealed that anti-P levels increased before and during the active phases of psychosis but not during sepsis or other exacerbations of SLE, and that the elevations were selective for anti-P antibodies, as opposed to anti-DNA antibodies. Longitudinal studies of anti-P activity in two patients with anti-P but without psychosis showed less than threefold changes in anti-P levels despite exacerbations of disease. We conclude that anti-P is associated with lupus psychosis and that synthetic peptide antigens may be useful for the detection and measurement of autoantibodies to intracellular proteins.

The characteristics of eukaryotic ribosomal proteins PO, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE). PO, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the-presence of 6 M urea. The apparent molecular size of the complex was 140 kDa as determined by gel fitration on a Sephadex G-200 column. PO may, therefore, be the eukaryotic equivalent ofEscherichia coli ribosomal protein L10. In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and PO and P1 were found to be more acidic than their ribosome-bound counterparts. Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia saUna (eL12). Sixteen SLE sera containing antibodies to PO, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 anmno acids. Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins. These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).Systemic lupus erythematosus (SLE) is an autoimmune disease generally characterized by serum antibodies directed against nuclear proteins and nucleic acids (1). That some patients' sera contain antibodies against ribosomal constituents (2, 3) has long been known, but the identity of these ribosomal antigens has only recently been determined (4). SLE anti-ribosome antibodies show almost exclusive reactivity against three 60S ribosomal subunit phosphoproteins called PO, P1, and P2 (4, 5). These same three proteins are also recognized by a mouse monoclonal antibody raised against chicken ribosomes (6). These "P" proteins have molecular sizes of -38, 19, and 17 kDa, respectively. P1 and P2 are believed to be the eukaryotic equivalent of the Escherichia coli ribosomal protein L12 and have been shown to contain sequences that are highly conserved among eukaryotes (6). Thus, P2 (7) from rat liver shows a high degree of amino acid sequence homology with Artemia salina ribosomal protein eL12 (8) and yeast ribosomal protein YPA1 (9). P1 and P2 also appear to be the functional counterparts of Artemia ribosomal proteins eL12' and eL12 and yeast ribosomal proteins YPA1/YPA2 (10-12). In order to further evaluate the remarkable specificity of the SLE anti-PO, -P1, and -P2 (anti-P) antibodies, we have mapped the antigenic determinant on the P proteins. This determinant is present on all three proteins and is contained within a common sequence of 22 amino acids at the carboxyl terminus of the Artemia r...

In Escherichia coli, the ability to elicit a heat shock response depends on the htpR gene product. Previous work has shown that the HtpR protein serves as a sigma factor (if32) for RNA polymerase that specifically recognizes heat shock promoters (A. D. Grossman, J. W. Erickson, and C. A. Gross Cell 38:383-390, 1984). In the present study we showed that g32 synthesized in vitro could stimulate the expression of heat shock genes. The in vitro-synthesized &2 was found to be associated with RNA polymerase. In vivo-synthesized 32 was also associated with RNA polymerase, and this polymerase (Eor32) could be isolated free of the standard polymerase (Ef70). E&2 was more active than Eof70 with heat shock genes; however, non-heat-shock genes were not transcribed by E&32. The in vitro expression of the htpR gene required E{J70 but did not require Eff32.

ABSTIRACT S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 450C express HS genes in vitro =8 times better than extracts from cells at 33TC. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% ofthat seen in extracts from cells at 330C. These results correlate well with the levels of HS ar factor and normal ca factor bound to RNA polymeraswe. Thus, there was an 8-fold increase in the HS or factor and a 60% decrease in the normal or factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS cv factor could be accounted for by a 3-fold increase in the level of HS or factor mRNA during heat induction.There is now considerable information available on the heat shock (HS) response in Escherichia coli. A temperature shift from -30'C to 40-450C results in the increased synthesis of at least 17 proteins (1), and this response is regulated by a 32-kDa protein, the product of the htpR gene (2-4). Based on sequence data that showed that the htpR gene product possessed a high degree of homology with the 70-kDa a-factor (cr70), it was predicted (5) that this protein functioned as a oa factor (o&32). This has been clearly demonstrated by Grossman et al. (6) who showed that the HS RNA polymerase (Eo-32) could recognize specific HS sequences present on HS genes (7). Our laboratory has demonstrated that the htpR gene on a plasmid template could be expressed in vitro by the normal cellular RNA polymerase (Eo-70) to yield &2, which was purified in the form of Eo2 (8). In addition, it was shown that the Eo-32 produced in vitro (and in vivo) preferentially stimulated the expression of HS genes such as dnaK and groEL.A major unanswered question relative to the HS response is how heat alters the level or activity of &r32 in cells. The present studies were initiated to determine whether changes in &32 levels during HS correlated with the expression of HS genes in vitro. MATERIALS AND METHODSPlasmids and E. coli Strains. Plasmid pKT200, which contains groEL, and plasmid pCG203, which contains the dnaK gene, were kindly supplied by C. Georgopoulos (University of Utah). Plasmid pFN97 containing the a-32 gene (4) was kindly supplied by F. Neidhardt (University of Michigan). Purified plasmid DNAs were obtained from cleared lysates after two successive centrifugations through CsCl/ ethidium bromide. A .32 mutant strain of E. coli (JC7623-htpR::TnS) was constructed in the laboratory of Graham C. Walker (Massachusetts Institute of Technology) and kindly supplied to us by F. Neidhardt. E. coli B cells were grown in 2 liters of LB (Luria-Bertani) medium (9) in an airlift fermenter (Bethesda Research Laboratories). Cells were grown to midlogarithmic phase at 330C and shifted first to 40'C and then to 450C. Each shift took -4 min, and once the desired temperature was reached, the cells were immedia...

Autoantibodies to three eukaryotic 60S ribosomal phosphoproteins P0, P1 and P2 have been found in the sera of 10–20% of patients with systemic lupus erythematosus (SLE). These three proteins share a common epitope contained within the carboxy terminal 22 amino acids of each protein. Because central nervous system disturbances, with major behavioural disorders, occur in a significant fraction of SLE patients, the antiribosomal autoantibodies were measured in this subset of SLE individuals to determine whether or not there was an association. This antibody is present in 90% of SLE patients who were diagnosed as having psychosis, secondary to the disease.