Manuela Ortiz

A novel centromere monospecific serum to a human autoepitope on the histone H3-like protein CENP-A

Centromere autoantibodies are commonly found in the serum of patients with some systemic autoimmune diseases. Previous studies have shown that a major human centromere autoantigen is the histone H3‐like protein CENP‐A. Although the human cDNA has been cloned, native CENP‐A has been neither isolated nor expressed in Escherichia coli, and specific antibodies to this chromatin‐associated centromere protein are not available yet. In this report, a highly charged peptide on CENP‐A (residues 3–17) was used to generate a monospecific antibody that reacts by immunoblots with the 17 kDa centromeric protein. Immunofluorescence analysis showed reactivity of this anti‐CENP‐A serum in several but not all mammalian culture cells analyzed, suggesting that the sequence of this histone‐like centromere protein could be more variable throughout evolution than originally thought. Selective extractions of human placenta nuclear proteins and immunoblot analysis indicated that CENP‐A behaves in a similar way to the core histone polypeptides after nuclease digestion of chromatin. Also, immunoblot analysis demonstrated that the CENP‐A peptide used as immunogen is a target region on the CENP‐A molecule in several but not all CREST patients analyzed with high titers of autoantibodies to the centromere. Lastly, we found that in Jurkat cells induced to apoptosis, CENP‐A remains associated with the centromere, in contrast to other human autoantigens studied during apoptosis.

CENPA a Genomic Marker for Centromere Activity and Human Diseases

Inheritance of genetic material requires that chromosomes segregate faithfully during cell division. Failure in this process can drive to aneuploidy phenomenon. Kinetochores are unique centromere macromolecular protein structures that attach chromosomes to the spindle for a proper movement and segregation. A unique type of nucleosomes of centromeric chromatin provides the base for kinetochore formation. A specific histone H3 variant, CENPA, replaces conventional histone H3 and together with centromere-specific-DNA-binding factors directs the assembly of active kinetochores. Recent studies on CENPA nucleosomal structure, epigenetic inheritance of centromeric chromatin and transcription of pericentric heterochromatin provide new clues to our understanding of centromere structure and function. This review highlights the role and dynamics of CENPA assembly into centromeres and the potential contribution of this kinetochore protein to autoimmune and cancer diseases in humans.

Aneugenic effects of benzoxazinones in cultured human cells.

Benzoxazinones (BAs) are natural products that are present in Gramineae and represent part of the plant defence system against pests. In recent years, sprouts of maize, wheat and rye have been used for the production of dietary supplements. We have investigated the potential genotoxic activities of a diverse range of synthetic derivatives of the most abundant natural BA, namely DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one), proposed for use as a potential herbicide. We have tested 18 synthetic BAs for potential effects in cultured HeLa cells. We found significantly higher micronucleus (MN) induction over the background level, with the solvent DMSO used as an internal control. Concentration-dependent effects were found between 1nM and 20nM for all the synthetic compounds studied. Immunostaining with an anticentromere antibody showed that >80% of MN induced gave a centromere-positive signal. Similarly, fluorescence in situ hybridization (FISH) analysis with alphoid centromere probes showed a positive hybridization signal, indicating that all compounds analyzed are aneugenic. Chemical modification of the N in the heterocyclic aromatic amine served us to suggest a relationship between the structure and the aneugenic effects of the compounds analyzed. Our findings indicate that benzoxazinoids could be potential genotoxins for human cells.

Anti-CENPI autoantibodies in scleroderma patients with features of autoimmune liver diseases

BACKGROUND Anticentromere autoantibodies have been reported to be associated with scleroderma and serve as a marker in different rheumatic diseases in humans. Major centromere autoantigens described so far include constitutive kinetochore proteins such as CENPA, CENPB, CENPC and CENPH and facultative proteins such as CENPE, CENPF and INCENP. We examined the inner kinetochore component CENPI as a new putative centromere autoantigen in scleroderma patients. METHODS To test for the presence of CENPI centromere autoantibodies, 72 sera from patients with systemic lupus erythematosus and systemic sclerosis were assayed by immunofluorescence and further tested by immunoblots with an Nt-CENPI recombinant protein. RESULTS 8 out of 31 (25.8%) patients diagnosed of scleroderma or Undifferentiated Connective Tissue Disease (UCTD) produced anti-CENPI autoantibodies. Epitopes were demonstrated to be located mainly but not exclusively in the N-terminal domain of the human CENPI protein. Five of the 8 (62.5%) CENPI positive sera also had other autoantibodies related to primary biliary cirrhosis. Further, two patients (25%) with anti-CENPI autoantibodies had concurrent diagnosis of primary biliary cirrhosis. CONCLUSIONS This study demonstrates that CENPI, a centromere protein that localizes to the inner kinetochore structure, is a human autoantigen. The significance of anti-CENPI autoantibodies could be relevant in scleroderma patients as a marker for concurrent autoimmune liver disease.

Autoantibodies against the chromosomal passenger protein INCENP found in a patient with Graham Little-Piccardi-Lassueur syndrome

BackgroundGraham Little – Piccardi – Lassueur (GLPL) syndrome is a rare dermatosis characterized by scarring alopecia, loss of pubic and axillary hair, and progressive development of variously located follicular papules. We report a first case ever of an autoimmune response in a patient suffering from GLPL syndrome.MethodsImmunofluorescence and immunoblot analysis were used in a variety of cell cultures including human, monkey, hamster, mouse and bovine cells to analyze the presence of autoantibodies in a GLPL patient.ResultsThe autoimmune serum showed a pattern of centromere and spindle microtubule staining resembling that of the chromosomal passenger protein complex. By using a complex of proteins expressed in baculovirus, immunoblot analysis demonstrated that the INCENP protein is a major autoantigen in this patient with GLPL syndrome.ConclusionAn autoimmune response in GLPL syndrome is reported against the INCENP centromere protein. The occasional development of autoimmunity in GLPL patients could serve as a test in continuing efforts to detect this disease and for a more directed therapy based on the autoantigen response.

Immunohistochemical detection of ribosomal transcription factor UBF: diagnostic value in malignant specimens.

The nucleolar organizer regions (NORs) of human chromosomes can be identified in interphase and mitotic cells by localization of some intrinsic components such as the associated enzyme RNA polymerase I. A new sensitive staining method for NORs is described using a specific antibody to the ribosomal transcription factor UBF. By indirect immunofluorescence and enzyme‐labelling methods, NORs stained in benign and malignant cells from a variety of tissues with monospecific anti‐UBF serum showed significant morphological differences which correlated well with histopathological evaluation. The number of NORs per cell in malignant preparations increased significantly. Furthermore, the staining of a NOR protein component such as UBF appears to be as sensitive as the silver‐staining technique (AgNOR) and might be a better alternative for detecting ribosomal activity in malignant tissues.