Biomedical subjects

N Pourmand

Publications and source records attributed to N Pourmand.

5 recordsLinked to original sources

PubMed2001

Method enabling fast partial sequencing of cDNA clones.

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

Base Sequence↗

PubMed2000

Ro 52kD autoantibodies are detected in a subset of ANA-negative sera.

To define Ro 52kD, Ro 60kD, and La specificities of autoantibodies within ANA-negative sera, samples from 64 ANA-negative but SSA positive patients undergoing investigation due to suspected CTD were analysed, using recombinant antigens and synthetic peptides by immunoblotting and ELISA. The sera were selected from 4025 sera submitted for routine ANA analysis. Antibodies to Ro or La were detected in 42/64 sera (65%). Anti-Ro 52kD antibodies occurred most frequently and were present in 42/64 sera (65%). This was the only specificity of autoantibody detected in 18 sera. No patient had only anti-La or anti-Ro 60 antibodies. In total 18.64 patients (28%) had Ro 60 antibodies and 14/64 had anti-La antibodies (21%). Eight patients had antibodies reacting with all three antigens. We used the same set of sera to test the antigenicity of different regions of Ro 52kD represented by deletion clones and peptides derived from the Ro 52kD sequence. Out of 30 sera reacting with a recombinant deletion clone encompassing as residues 136-227, 12 sera reacted with a peptide corresponding to a 200-239. Some sera gave a low positive OD value with a peptide of a 176-196. Based on the results of this study in which we demonstrate Ro 52kD autoantibodies in 65% of selected ANA negative sera and define an autocephitope within the Ro 52kD protein composed of the leucine zipper domain, we suggest that testing for Ro 52kD antibodies could be included in an extended investigation of ANA negative patients with suspected connective tissue disease.

Antibodies, Antinuclear↗

PubMed1999

Ro/SSA and La/SSB specific IgA autoantibodies in serum of patients with Sjögren's syndrome and systemic lupus erythematosus.

OBJECTIVE: To investigate the occurrence of IgA autoantibodies to Ro 52 kDa, Ro 60 kDa and La antigen in serum of patients with primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE). METHODS: Recombinant Ro 52 kDa, Ro 60 kDa and La antigens were used to analyse autoantibodies in serum from 25 patients with pSS, 30 patients with SLE and 20 controls using a semiquantitative immunoblotting approach. RESULTS: Among the patients with pSS, 21 (84%) had detectable IgA autoantibodies to Ro 52 kDa, 13 (52%) to Ro 60 kDa and 20 (80%) to La antigen. The corresponding results for the patients with SLE were 22 (73%), 14 (47%) and 20 (67%), respectively. No IgA autoantibodies against the three antigens were detected in 20 normal controls. A comparison of several clinical features with the titres of IgA antibodies to Ro 52 kDa, Ro 60 kDa and La, revealed a significant relation between IgA anti-Ro 52 and IgA anti-La to sicca (p< 0.05). Semiquantitative data suggest that IgG is the dominating antibody to the three antigens followed by IgM > IgA in both SLE and pSS patients. Specificity studies of IgA autoantibodies with different subfragments of Ro 52 kDa and Ro 60 kDa antigens showed that IgA antibodies did not differ from IgG and IgM in their recognition pattern. CONCLUSION: These results suggest that besides IgM and IgG, IgA autoantibodies are also detected at high frequency in patients with pSS and SLE. Further studies are necessary to evaluate the contribution of these IgA autoantibodies to inflammation as well as their diagnostic value.

Adult↗

PubMed1998

The Zn2+ binding domain of the human Ro 52 kDa protein is a target for conformation-dependent autoantibodies.

The Ro 52 kDa protein was originally identified using autoimmune sera directed against the Ro/SS-A antigen, also known as Ro ribonucleoprotein particles (Ro RNPs). In most human cells these consist of one of four small human cytoplasmic (hY) RNA molecules complexed with the Ro 60 kDa protein. This protein interacts directly with the hY RNAs, whereas the Ro 52 kDa protein is thought to associate via protein-protein interactions. The Ro RNPs are present in all mammalian cells, but their intracellular location and function remain unclear. The primary structure shows that the Ro 52 kDa protein is a member of a small family of proteins sharing two features, a leucine zipper region and an aminoterminal cysteine-histidine rich region with two different putative zinc finger motifs. To study if the Ro 52 kDa protein actually binds Zn2+, both the full-length Ro 52 kDa clone and various subclones were expressed as recombinant proteins and assayed for Zn2+ binding in vitro. A fragment containing the first cysteine-histidine cluster at residues 14-54 and other larger overlapping fragments were found to bind Zn2+. In this report we also demonstrate that the Zn2+ binding domain is a target for conformation-dependent anti-Ro 52 kDa autoantibodies. The antigenicity is dramatically increased by the same reducing conditions that promote Zn2+ binding. This is in contrast to the previously described immunodominant Ro 52 kDa domain.

Autoantibodies↗

PubMed1998

Intracellular localisation of the Ro 52kD auto-antigen in HeLa cells visualised with green fluorescent protein chimeras.

Autoantibodies to the Ro/SSA and La/SSB antigens are found in patients with Sjogren's syndrome and systemic lupus erythematosus. The Ro/SSA autoantigen consists of a 52 kD and a 60 kD protein, complexed with one of four small RNA molecules. The La protein can associate with the complex. The Ro/SSA autoantigens are present in all mammalian cells, but their intracellular location is subject of controversy and their function remains unclear. To study the intracellular sorting and targeting of Ro 52 kD we have constructed expression plasmids encoding fusion proteins between the full-length Ro 52 kD protein as well as Ro 52 kD fragments and the green fluorescent protein (GFP) from the jelly fish, Aequorea Victoria. The subcellular distribution of the GFP-Ro 52 kD fusion proteins was investigated in transient expression experiments using transfected HeLa cells. The GFP-full-length Ro 52 kD fusion protein was accumulated in the cytoplasm and excluded from the nucleus. When GFP was fused with the La protein, the fluorescence was located in the nucleus. Clones coding for Ro 52 kD fragments containing the hydrophilic central part of the Ro 52 kD protein gave the same intracellular location and type of cytoplasmic speckles as the full-length Ro 52 kD protein. In contrast, both amino terminal and carboxy terminal fragments were uniformly distributed throughout the cell just like the GFP protein itself. These observations indicated a cytoplasmic location of the Ro 52 kD protein and demonstrated the crucial role of the hydrophilic domain in restricting the Ro 52 kD protein to this intracellular compartment.

Animals↗