A comparative study of the chemical stability of various mitomycin C solutions used in glaucoma filtering surgery.

OBJECTIVE: To determine the chemical stability of various mitomycin C (MMC) solutions used in glaucoma filtering surgery. METHODS: A survey of the MMC solutions currently in use in 21 hospitals (11 in Canada, 10 in the United States) was conducted. A comparative study of the chemical stability of five different representative solutions was performed. The effects of buffer and storage variables on the chemical breakdown of MMC in the solutions were studied by means of high-performance liquid chromatography (HPLC). RESULTS: The survey revealed 33 different variations (including recipes and storage conditions) in the preparation of MMC solutions. Although the majority of the hospitals (15 of 21; 72%) were preparing stable solutions, six of the hospitals (28%) were preparing potentially unstable solutions. The stability of the solutions varied in a nonuniform manner when stored at different temperatures in different buffers. CONCLUSION: The lack of standardization and quality control of MMC solutions used in filtering surgery allows for the possibility of hospitals preparing unstable solutions.

Tissue-specific expression in mammalian brain, heart, and muscle of S1, a member of the elongation factor-1 alpha gene family.

Elongation factor-1 alpha (EF-1 alpha) is an ubiquitous protein that functions in peptide elongation during mRNA translation. We previously reported the isolation of a rat S1 protein that is antigenically related to statin, a nonproliferation-specific protein; this S1 gene shares a high degree of homology to EF-1 alpha. We constructed specific riboprobes to the two genes, based on the difference in the 3' noncoding regions of both S1 and EF-1 alpha mRNAs. Northern analysis and RNase protection assays have revealed that S1 mRNA is present only in brain, heart, and muscle, while EF-1 alpha mRNA has been detected in all tissues surveyed so far. The same tissue specificity has been observed in mouse, suggesting that S1 expression is conserved between these two mammalian species. S1 transcript was detected in late brain embryogenesis (day 20), but in lower amounts than in 3-month-old adult brain. We show that the relative levels of both S1 and EF-1 alpha transcripts and their respective tissue abundances remain unchanged during the aging process. The function of S1 is not yet known; but these results suggest that it may be involved in specific control mechanisms for protein synthesis in tissues where cells (i.e. neurons and myocytes) are permanently locked in a state of nonproliferation.

Anti-SM and anti-U1-RNP lupus antibody fine specificities.

Evidence for the presence of multiple shared epitopes on the target cellular antigens was found when 290 anti-Sm and anti-U1-RNP lupus sera were analyzed by immunoblotting. Forty-eight different immunoblot patterns were observed with the sera. Studies with selected antigen-affinity-purified antibodies confirmed the presence of multiple shared epitopes, in agreement with findings obtained with monoclonal antibodies. The results have implications for the design of effective diagnostics and for the use of these antibodies as molecular probes.

Analysis of autoantibody specificities in selected systemic lupus erythematosus (SLE) sera.

In order to design effective diagnostics for lupus, the heterogeneity in patient response must be understood. This heterogeneity in the anti-Sm and anti-U1-RNP response was examined via a frequency analysis of autoantibody fine specificities. Thus, 275 sera were studied by immunoprecipitation, immunoblotting, and immunodiffusion, and the frequency of occurrence of different autoantibodies to individual snRNP polypeptides and to other HeLa cell polypeptides was determined. The sera were found to contain autoantibodies reactive with denatured as well as native forms of HeLa-cell polypeptides. The common occurrence of several novel antibody fine specificities was noted, such as anti-p45 (different from anti-La/SS-B), anti-p105, and anti-p115. Another group of autoantibodies that is apparently not disease associated was observed in both lupus and normal sera.

The isolation of interferon-inducing mutants of vesicular stomatitis virus with altered viral P function for the inhibition of total protein synthesis.

We have previously reported that T1026, a temperature-sensitive (ts) noncytocidal mutant of VSV, and its ts revertant, T1026-R1, are nonconditional mutants in the VSV function "P" for the inhibition of total protein synthesis (viral plus cellular) in infected cells (C. P. Stanners, A. M. Francoeur, and T. Lam, 1977, Cell 11, 273-281; C. P. Stanners, S. Kennedy, and L. Poliquin, 1987, Virology 160, 255-258). We have also shown that P- mutants such as these are superior interferon inducers relative to their parental P+ wild-type virus, HR, and that P- mutants may be distinguished from P+ virus using the plaque interferon production of PIF assay. (A. M. Francoeur, T. Lam, and C. P. Stanners, 1980, Virology 105, 526-536). In order to carry the analysis of VSV P function further, a number of independent mutants in the VSV P function are required. We show here that the PIF assay may be used to isolate spontaneously occurring interferon-inducing mutants (PIF+ mutants) from wild-type VSV (PIF- virus) populations. About one-half of the PIF+ mutants isolated with the PIF assay were found to have alterations in the VSV P function. As well as mutants that were defective for the inhibition of total protein synthesis, the assay yielded a new class of VSV P function mutants which appear to inhibit protein synthesis more severely than does P+ virus. The majority of newly isolated PIF+ mutants was also found to be temperature sensitive for growth. The ts phenotype, however, could be reverted for most PIF+ mutants with little effect on the PIF or P phenotype. These findings show that interferon induction and P function are related functions of VSV; this fact has allowed the isolation of a repertoire of mutants with widely varying P function.

Epitopes, structural domains, and asymmetry of amino acid residues in SS-B/La nuclear protein.

SS-B/La is a conserved cellular phosphoprotein of 46 to 48 KD that is the target antigen of autoantibodies in sera of patients with Sjogren's syndrome and systemic lupus erythematosus. SS-B/La is also known to be associated with certain small cellular and viral RNA, including adenovirus VAI and VAII RNA. Two relatively protease-resistant domains (X and Y) were defined in SS-B from HeLa cells by using human autoantibodies as reagents. Domain X, a methionine-containing nonphosphorylated 28 KD polypeptide, was found to be resistant to partial digestion with six different proteases. Similar domains were also found in calf and rabbit SS-B. Domain Y, a 23 KD polypeptide, was detected after limited digestion with S. aureus V8 and trypsin. This domain contained little if any methionine, but all the detectable phosphorylated amino acids. Among 16 anti-SS-B sera tested by immunoblotting, 11 (69%) were reactive with both domains, three (19%) only with domain X, and two (13%) only with domain Y. These results showed that there are at least two distinct antigenic epitopes on the 46 to 48 KD SS-B/La protein, each located on a separate structural domain. The asymmetric distribution of methionine and phosphorylated amino acid residues in SS-B/La show striking similarity to the two reported domains of the adenovirus 72 KD DNA-binding protein, and raises questions concerning functional similarities that await investigation.

Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity.

Anti-Ki (Ku, p70/p80) autoantibodies, named after the prototype patient Kikuta by Tojo et al., occur in approximately 10% of patients with SLE, often in association with anti-Sm autoantibodies. The immunofluorescent staining pattern characteristic of anti-Ki antibodies is diffuse speckled nuclear, although some substrates show nucleolar staining as well. Anti-Ki sera specifically immunoprecipitated two protein antigens, Ki86 (Mr 86,000) and Ki66 (Mr 66,000), from radiolabeled cell extracts. The Ki system was found to be immunologically identical to the Ku system described by Mimori et al. and the p70/p80 system described by Reeves. The Ki primary in vitro translation products were identified and proved similar in size to the cellular antigens. The Ki antigens were purified from human spleen by immunoaffinity chromatography followed by SDS-PAGE. The purified Ki antigens proved to be closely related by amino acid composition, and did not appear to be phosphorylated, glycosylated, or associated with RNA. The Ki antigens were found to bind to DNA, in agreement with the observations on the Ku and p70/p80 antigens. They were found to be widely conserved in mammals and were coordinately expressed in all tissues tested. Anti-Ki autoantibodies were purified by antigen-affinity chromatography and were tested by immunoblotting. The antibodies were classified as class I, II, or III, depending on their reactivity with the Ki antigens in immunoblots. Class I antibodies cross-reacted with both Ki antigens, class II antibodies reacted solely with Ki66, and class III antibodies reacted solely with Ki86. These results suggest that at least three different epitopes are present on the Ki autoantigens and that patients differ in their autoantibody response to each epitope.

Identification of ribosomal protein autoantigens.

Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome. We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting. The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria. Thus the human autoantibodies recognize epitopes that are widely conserved in evolution. Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast). These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E. coli. The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit. The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins. In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA. These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.

Synthesis of small nuclear ribonucleoprotein particles by the malarial parasite Plasmodium falciparum.

Sera from patients with autoimmune diseases have been used to identify small nuclear ribonucleoprotein particles (snRNPs) present in higher eukaryotic cells and also in dinoflagellates. Previously these sera have not detected crossreactive snRNP protein antigens of other lower eukaryotes such as yeast, Tetrahymena, or Dictyostelium. We report that anti-Sm, anti-U1-RNP, and anti-La/SS-B human antisera react with specific snRNP protein antigens synthesized by the protozoan Plasmodium falciparum, the human malarial parasite. These results suggest that the structure and antigenicity (and thus probably the function) of snRNPs have been widely conserved in eukaryote evolution.

Characterization and purification of lupus antigen La, and RNA-binding protein.

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.

La antigen recognizes and binds to the 3'-oligouridylate tail of a small RNA.

The La antigen is a cellular protein which interacts with many RNA species that are products of RNA polymerase III, including the adenovirus virus-associated (VA) RNAs. We demonstrate that the efficiency of antigen binding in vitro is determined by the number of U residues at the RNA 3' terminus. Forms of VA RNAI with more than two terminal U residues are fully bound, forms with two U residues are partially bound, and forms with fewer than two U residues are not bound at all. The antigen can be covalently linked to VA RNA by UV irradiation, and the site of cross-linking is shown to contain the 3' terminus of the RNA. We conclude that the antigen recognizes the U-rich 3' tail of VA RNA, and presumably that of other polymerase III products, and that it binds at or close to this site.

Cellular protein and RNA antigens in autoimmune disease.

Antibodies directed against soluble cellular antigens are a distinctive feature of systemic autoimmune disease. We have examined 22 autoantibodies in sera from 1111 patients and present the disease associations together with a biochemical analysis of the antigens. The data emphasize the clinical specificity of the antibodies and the restricted number of cellular components that commonly elicit an immune response. In several instances, serological relationships between antibodies mirror biochemical relationships between the corresponding antigens. The antigens are mainly proteins and are often present in complexes with additional protein or nucleic acid molecules. In myositis the antibodies react chiefly with cytoplasmic antigens such as aminoacyl-tRNA synthetases, in contrast to the mainly antinuclear response in SLE. It is argued that both environmental stimuli and genetic factors govern autoantibody specificity, and that molecular characterization of the cellular antigens may yield clues to the aetiology of the disease and of the concomitant, specific autoimmune response.

Interaction between VA RNA and the lupus antigen La: formation of a ribonucleoprotein particle in vitro.

The small adenovirus-encoded VA RNAs occur as ribonucleoprotein (RNP) particles in association with a cellular protein antigen, La, recognized by the anti-La class of lupus sera [Lerner, M. R., Boyle, J. A., Hardin, J. A. & Steitz, J. A. (1981) Science 211, 400-402]. We have tentatively identified the La antigen as a HeLa cell phosphoprotein of Mr approximately equal to 45,000, present in infected and uninfected cells. The antigen appears not to be required for the transcription of VA RNAs in vitro. RNP particles that contain newly synthesized VA RNAs assemble rapidly in transcription extracts making VA RNA and also can be reconstituted from purified VA RNA and a source of La antigen. Variant forms of VA RNAI with sequence deletions and substitutions bind to the La antigen, suggesting that the recognition site includes the RNA termini or the sequences corresponding to the internal control region (promoter), or both. Upon reconstitution with fragments of VA RNAI, oligonucleotides from both the 5' and 3' termini bind to the antigen, but those from the control region do not. The terminal oligonucleotides of wild-type VA RNA can form a basepaired stem, but structures of comparable stability cannot be formed by the chimeric variant molecules. Therefore, the recognition site is probably the terminal nucleotides themselves rather than the stem structure.

Cellular quiescence modulates and replication of L15 mutants of vesicular stomatitis virus.

Infectious particle production by temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) was measured in a variety of different host cell types maintained in a state of quiescence or stimulated to proliferate. At permissive temperatures, all ts mutants and the wild-type virus replicated equally well and with the same kinetics in both quiescent and proliferating cells. At semi-permissive temperatures, however, Lts mutants, with temperature-sensitive virion polymerases, showed a delay of about 6 h in infectious particle production relative to wild-type virus in proliferating cells and greater than 16 h in quiescent cells. The effect was specific for the Lts class of mutants and was not seen for representative mutants in any of the four other complementation groups of VSV. Regarding cellular determinants, the effect was correlated only with the growth phase and not with the species of origin, interferon inducibility or with malignant transformation.

Analysis of VSV mutant with attenuated cytopathogenicity: mutation in viral function, P, for inhibition of protein synthesis.

T1026, a ts mutant of VSV which is much less cytopathogenic than its parent, HR, and which can establish persistent infection under certain conditions, is a double mutant. In addition to its ts mutation in the virion RNA polymerase, T1026 has a second non-ts mutation in a viral function termed "P". This function is responsible for the inhibition of total protein synthesis in infected cells and acts chiefly at the level of translational initiation. In some cell systems, the inhibition of protein synthesis produced by P appears to be selective for cellular protein synthesis, whereas in other cell systems, both cellular and viral protein synthesis are inhibited. T1026 and its ts revertants are phenotypically P- -that is, cells infected with them show total protein synthesis rates equal to or greater than uninfected cells, while synthesizing viral proteins at the same or even greater rates than HR-infected cells. The P- mutation is correlated with failure to increase plaque size after 2-3 days of incubation. Since viral mutants obtained from persistently infected cultures in a variety of systems appear to be double mutants with a ts mutation in the virion RNA polymerase and a small plaque marker, we suggest that T1026 could represent a model for such mutants.