Cross-reactions between Leishmania braziliensis and Trypanosoma cruzi caused by common antigenic determinants hinder the specific diagnosis of cutaneous and mucocutaneous leishmaniasis (CL and MCL). Therefore, the usefulness of the 70-kDa heat shock protein (Hsp70) from L. braziliensis for differential serodiagnosis was investigated. The single-copy gene encoding Hsp70, consisting of 663 amino acids, was isolated from a genomic DNA library. The antigenicity data show that Hsp70 is an immunodominant antigen highly recognized (84%) by sera of patients with CL and MCL and to a lesser extent by chagasic patients (18.75%). Antigenic mapping of the 5 overlapping fragments into which the protein was split showed that the main antigenic determinants are located in the carboxy-terminal end. The linear antigenic determinants that show cross-reactions with chagasic sera are located in the fragment rLb70(352-518). The carboxy-terminal fragment rLb70(513-663) presents 70% sensitivity and 100% specificity, so it could be a potential candidate for specific serodiagnosis of CL and MCL caused by L. braziliensis.

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Synthesis of invertase (EC 3.2.1.26) in Pichia anomala is controlled by the carbon source in the culture medium. The enzyme was purified to homogeneity from P. anomala cells fully derepressed for invertase synthesis and shown to be a multimeric glycoprotein composed of identical subunits with an apparent molecular mass of 86.5 kDa. The carbohydrate moiety accounts for approx. 30% of the total mass of the molecule and consists of manno-oligosaccharides N-linked to the polypeptide. Most of the characteristics of the enzyme analysed in this study were similar to those previously reported for other yeast invertases, with the remarkable exception of its thermal sensitivity which appears after 15 min incubation at temperatures above 32 degrees C.

The characteristics of the Scl-70 antigen (topoisomerase I) have been analyzed by means of autoantibodies. This antigen is a DNA-binding protein, dissociable from DNA at 0.3M NaCl and bound to a fraction of DNA that is very sensitive to nucleases. The molecular weight of the antigen is 105,000 daltons, whether dissociation conditions are used or not. Using chicken erythrocytes, and taking advantage of the strong interaction of the antigen with hydroxyapatite, we have designed a simple and fast purification protocol that allows the determination of anti-topoisomerase I antibodies by enzyme-linked immunosorbent assay.The presence of autoantibodies against nuclear structures is characteristic of many autoimmune diseases (1). Nevertheless, it is not yet known why these autoantibodies are produced, or what role they play in the pathologic process.Approximately 20% of patients with scleroderma have autoantibodies to a nuclear antigen designated the Scl-70 antigen (2). This antigen was first defined as a 70-kd chromatin antigen (3), but since then, it has been postulated that it could be an 86-kd By means of autoantibodies, we have analyzed the molecular characteristics of the Scl-70 antigen, the type of interaction with DNA, and the topoisomerase activity. A fast purification protocol, which allows the quantification of anti-Scl-70 antibodies by enzymelinked immunosorbent assay (ELISA), is also described. MATERIALS AND METHODSSera. Sera were collected from 9 patients who had been diagnosed as having scleroderma (7). In 5 of these sera, anti-Scl-70 antibodies were detected by immunodiffusion, using reference sera obtained from Dr. Eng Tan (Scripps Clinic, La Jolla, CA) as control. The patients' sera were able to bind a 70-kd protein from an extract obtained as described by Douvas et al (3). In addition, sera with antinuclear antibodies from 10 patients (4 with systemic lupus erythematosus, 3 with mixed connective tissue disease, 2 with Sjogren's syndrome, and 1 with rheumatoid arthritis) and 9 normal human sera were used in blotting and ELISA. Finally, anti-DNA and anti-Sm antibodies were used as controls in the immunofluorescence studies.Immunofluorescence. Indirect immunofluorescence studies were performed with cytocentrifuged cells or nuclei and with cells grown on coverslips, using human melanoma cell lines HeLa and HMcB. Cells were fixed with acetone for 5 minutes or made permeable by freezekhawing and treatment with 0.5% NP-40 for 5 minutes. Afterwards, cells or nuclei were treated for 30 minutes with: (a) phosphate buffered saline (PBS) as a control; (b) 10 mM phosphate buffer, pH 7.2, containing 0.3,0.5, 1 , or 2M NaCl; (c) DNase

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine-lacking medium, indicating that it encodes a product able to complement the b-isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC-rich palindrome motif identified in the 5k region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3k-terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1-PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation. The nucleotide sequence has been submitted to the EMBL database under Accession No. AJ294714.