Monoclonal antibody against carcinoembryonic antigen (CEA) identifies two new forms of crossreacting antigens of molecular weight 90,000 and 160,000 in normal granulocytes.

Two new forms of non-specific crossreacting antigens (NCAs) were identified in the Nonidet P40 (NP-40) extracts of normal granulocytes by precipitation with the monoclonal antibody (MAb) 192 directed against carcinoembryonic antigen (CEA) and already known to crossreact with the perchloric acid soluble NCA-55. The NP-40 soluble NCAs recognized by MAb 192 have apparent mol. wts of 90,000 and 160,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both NCAs appear to consist of a single monomeric polypeptide chain, since they have the same electrophoretic mobility in SDS-PAGE under reduced and non-reduced conditions. When granulocytes were extracted with perchloric acid instead of NP-40, only the 55,000 mol. wt antigen, corresponding to the previously described NCA-55, was precipitated by MAb 192. Furthermore, it was shown that NCA-55 is not a degradation product of NCA-90 or NCA-160 due to the perchloric acid treatment because exposure to perchloric acid of NCA preparations purified from NP-40 extracts did not change their apparent mol. wts in SDS-PAGE. It was also shown that NCA-160 is not a granulocytic form of CEA because it was not precipitated by the MAb 35 reacting exclusively with CEA. Immunocytochemical studies of granulocytes and macrophages showed that MAb 192 stained both types of cells whereas MAb 47 stained only the granulocytes and MAb 35 none of these cells. In granulocytes both MAbs reacted with antigens associated with granules and also present at the periphery of the nucleus as well as in the Golgi apparatus. The NCA-90 identified by MAb 192 was found by sequential immunodepletion to be antigenically distinct from the NCA-95 precipitated by MAb 47. The epitope recognized by MAb 192 on CEA and NCA molecules appears to be on the peptidic moiety because the antigens deglycosylated by the enzyme Endo F were still precipitated by this MAb. Taken together, the results indicate that MAb 192 identifies two novel forms of NCA (NCA-90 and NCA-160) in NP-40 extracts of granulocytes, which are distinct from CEA and the previously described NCA-55 and NCA-95 identified by MAbs 192 and 47, respectively, in perchloric acid extracts of granulocytes.

Two functional forms of the Xenopus laevis estrogen receptor translated from a single mRNA species.

Steroid receptors are nuclear proteins that regulate gene transcription in a ligand-dependent manner. Over-expression of the Xenopus estrogen receptor in a vaccinia virus-derived expression system revealed that the receptor localized exclusively in the nucleus of the infected cells, irrespective of the presence or absence of the ligand. Furthermore, two forms of the receptor were produced, a full-length and a N-terminal truncated version, which are translated from a single mRNA species by the use of two AUG within the same reading frame. These 66- and 61-kDa receptors were also observed after in vitro translation of the mRNA as well as in primary Xenopus hepatocytes. Both forms are potent estrogen-dependent transcriptional activators in transient transfection experiments, as well as in in vitro transcription assays.

Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein.

Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.

Equivalence of paper and pencil vs internet forms of the ZKPQ-50-CC in Spanish and French samples

This study was designed to check for the equivalence of the ZKPQ-50-CC (Spanish and French versions) through Internet on-line (OL) and paper and pencil (PP) answer format. Differences in means and devia- tions were significant in some scales, but effect sizes are minimal except for Sociability in the Spanish sample. Alpha reliabilities are also very similar in both versions with no significant differences between formats. A robust factorial structure was found for the two formats and the average congruency coefficients were 0.98. The goodness-of-fit indexes obtained by confirmatory factorial analysis are very similar to those obtained in the ZKPQ-50-CC validation study and they do not differ between the two formats. The multi-group analysis confirms the equivalence among the OL-PP formats in both countries. These results in general support the validity and reliability of the Internet as a method in investigations using the ZKPQ-50-CC.

Regulation of nonphosphorylated and phosphorylated forms of neurofilament proteins in the prefrontal cortex of human opioid addicts.

The neurofilament (NF) proteins (NF-H, NF-M, and NF-L for high, medium, and low molecular weights) play a crucial role in the organization of neuronal shape and function. In a preliminary study, the abundance of total NF-L was shown to be decreased in brains of opioid addicts. Because of the potential relevance of NF abnormalities in opioid addiction, we quantitated nonphosphorylated and phosphorylated NF in postmortem brains from 12 well-defined opioid abusers who had died of an opiate overdose (heroin or methadone). Levels of NF were assessed by immunoblotting techniques using phospho-independent and phospho-dependent antibodies, and the relative (% changes in immunoreactivity) and absolute (changes in ng NF/microg total protein) amounts of NF were calculated. Decreased levels of nonphosphorylated NF-H (42-32%), NF-M (14-9%) and NF-L (30-29%) were found in the prefrontal cortex of opioid addicts compared with sex, age, and postmortem delay-matched controls. In contrast, increased levels of phosphorylated NF-H (58-41%) and NF-M (56-28%) were found in the same brains of opioid addicts. The ratio of phosphorylated to nonphosphorylated NF-H in opioid addicts (3.4) was greater than that in control subjects (1.6). In the same brains of opioid addicts, the levels of protein phosphatase of the type 2A were found unchanged, which indicated that the hyperphosphorylation of NF-H is not the result of a reduced dephosphorylation process. The immunodensities of GFAP (the specific glial cytoskeletol protein), alpha-internexin (a neuronal filament related to NF-L) and synaptophysin (a synapse-specific protein) were found unchanged, suggesting a lack of gross changes in glial reaction, other intermediate filaments of the neuronal cytoskeletol, and synaptic density in the prefrontal cortex of opioid addicts. These marked reductions in total NF proteins and the aberrant hyperphosphorylation of NF-H in brains of opioid addicts may play a significant role in the cellular mechanisms of opioid addiction.

Persisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis

BACKGROUND: The long latent stage seen in syphilis, followed by chronic central nervous system infection and inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponema pallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis. METHOD: Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, and exposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms may be induced in vitro, following infection of primary chicken and rat neurons, as well as rat and human astrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur in vivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods to detect evidence of neuroinflammation in the form of reactive microglia and astrocytes. RESULTS: Under these conditions we observed atypical cystic, rolled and granular forms of these spirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark field and atomic force microscopy (AFM) methods. The atypical and cystic forms found in the brains of three patients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced in vitro. We also observed nuclear fragmentation of the infected astrocytes using the TUNEL method. Abundant HLA-DR positive microglia and GFAP positive reactive astrocytes were present in the cerebral cortex. CONCLUSION: The results indicate that atypical extra- and intracellular pleomorphic and cystic forms of Borrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. The persistence of these more resistant spirochete forms, and their intracellular location in neurons and glial cells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest that Borrelia burgdorferi may induce cellular dysfunction and apoptosis. The detection and recognition of atypical, cystic and granular forms in infected tissues is essential for the diagnosis and the treatment as they can occur in the absence of the typical spiral Borrelia form.