Biomedical subjects

Ravi Kothapalli

Publications and source records attributed to Ravi Kothapalli.

6 recordsLinked to original sources

PubMed2005

Constitutive production of proinflammatory cytokines RANTES, MIP-1beta and IL-18 characterizes LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disease often associated with autoimmune disorders such as rheumatoid arthritis. High levels of soluble Fas ligand have been implicated in development of chronic neutropenia. However, a comprehensive analysis of constitutive chemokine and lymphokine production in LGL leukemia has not previously been reported. Here, we utilized RNase protection assays and enzyme-linked immunosorbent assays (ELISAs) to address this question. RANTES, IL-8, MIP-1alpha, MIP-1beta, IL-1beta, IL-10, IL-12 p35, IL-18, IFN-gamma and IL-1Ra were the cytokine transcripts expressed in elevated levels from RNA of peripheral blood mononuclear cells of LGL leukemia patients. Confirmatory ELISAs indicated that sera from LGL leukemia patients have elevated levels of RANTES, MIP-1beta, IL-18, and to a lesser extent IL-8 and IL-1Ra. This pattern of cytokine upregulation is similar to that seen in some chronic infections or in autoimmune diseases, thus characterizing LGL leukemia as a proinflammatory disorder.

Adult↗

PubMed2003

Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template.

In order to identify the reverse transcriptase activity in sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of large granular lymphocyte leukemia patients product enhanced reverse transcriptase activity (PERT) assays were performed using bacteriophage MS2 RNA as a template. All samples obtained from conditioned media of virus-infected cell lines as well as PBMCs of lymphocytic leukemia patients and normal healthy individuals tested positive with this assay. Therefore the validity of the assay was questioned. Careful evaluation of the assay revealed that some of the essential reagents used, such as Taq DNA polymerase and RNase inhibitor contained indigenous amplifiable DNA. DNase I treatment of Taq DNA polymerase before PCR reduced the product significantly. Moreover, no false positive results were observed when encephalomyocarditis virus RNA was used instead of MS2 RNA as the template. These results suggest a need for caution when using bacteriophage MS2 RNA as the template in PERT assays to confirm the presence of retroviral infection or for identification of novel retroviruses.

Humans↗

PubMed2003

Characterization of a variant of PAC-1 in large granular lymphocyte leukemia.

Phosphatase in activated T cells (PAC-1) is a mitogen-induced early responsive gene. It encodes a 32 kDa tyrosine-threonine dual specificity phosphatase. Constitutive expression of PAC-1 leads to an inhibition of MAP kinase activity in vivo. Such constitutive expression was reported in HTLV-1 infected cell lines. In the present study, we observed the constitutive over-expression of two transcripts related to PAC-1 in large granular lymphocyte (LGL) leukemia. By screening a LGL leukemia cDNA library using the 3' end of a PAC-1 probe, we obtained a clone (clone 8) which retains one and one half introns, excludes two exons, and matches one hundred percent with a DNA sequence on chromosome 2. The deduced amino acid sequence of the predicted protein contains 170 amino acids and is 144 amino acids shorter than PAC-1. When we expressed this protein in Escherichia coli as a GST-fusion protein, a 45 kDa (19 kDa PAC-1 variant+26 kDa GST protein) protein was obtained. The expressed protein was purified to near homogeneity by using a glutathione affinity column. The purified protein did not have any intrinsic phosphatase activity when assayed in vitro. But when this purified protein was added to a phosphatase assay system in combination with a recombinant dual specificity phosphatase, CL100, enhanced phosphatase activity was observed. The significance of the constitutive over-expression and its physiological role of this protein remain to be established in leukemic LGL.

Amino Acid Sequence↗

PubMed2003

Constitutive expression of cytotoxic proteases and down-regulation of protease inhibitors in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with rheumatoid arthritis. The etiology of LGL leukemia is not known. In order to better understand the pathogenesis of LGL leukemia, we analyzed differential gene expression using microarray technology. We found that approximately 80 genes were up-regulated and 12 genes were down-regulated when compared to normal peripheral blood mononuclear cells (PBMC). In the present study, we were interested in a group of genes involved in cytotoxic function. The up-regulated genes involved in cytotoxic function were serine proteinases (granzymes A, B, H and K) cysteine proteinases [cathepsin C, cathepsin W (lymphopain)], calpain small subunit and caspase-8. In addition, a pore-forming protein perforin, was also up-regulated. Northern blot analysis and RNase protection assays (RPA) confirmed that these genes were over-expressed in the majority of samples from LGL leukemia patients. Of interest, proteolytic inhibitors such as cystatin C, A, alpha-1 antitrypsin and metalloproteinase inhibitors were down-regulated in leukemic LGL when compared to normal peripheral blood mononuclear cells. Importantly, the pattern of gene expression in leukemic LGL resembles that seen in activated cytotoxic T cells (CTL).

Cathepsin C↗

PubMed2002

Characterization of a human sphingosine-1-phosphate receptor gene (S1P5) and its differential expression in LGL leukemia.

Large granular lymphocyte (LGL) leukemia is a lymphoproliferative disorder often associated with autoimmune disease. A central feature of this disease is dysregulation of apoptosis. In order to identify differentially expressed genes in LGL leukemia, microarray analysis was performed. We found many differentially expressed genes including several expression sequence tags (ESTs). As a systematic study, we selected one up-regulated EST (GenBank Accession number N47089) and further investigated. An LGL leukemia library was screened using this EST as a probe and a full-length sequence for a novel gene was identified. The deduced amino acid sequence revealed that the novel gene encodes a G-protein-coupled receptor gene that exhibits 86% identity with rat sphingosine-1-phosphate receptor (edg-8/nrg-1). This gene is present in brain, spleen, and peripheral blood mononuclear cells (PBMC) and is overexpressed in leukemic LGL.

Amino Acid Sequence↗

PubMed2002

Microarray results: how accurate are they?

BACKGROUND: DNA microarray technology is a powerful technique that was recently developed in order to analyze thousands of genes in a short time. Presently, microarrays, or chips, of the cDNA type and oligonucleotide type are available from several sources. The number of publications in this area is increasing exponentially. RESULTS: In this study, microarray data obtained from two different commercially available systems were critically evaluated. Our analysis revealed several inconsistencies in the data obtained from the two different microarrays. Problems encountered included inconsistent sequence fidelity of the spotted microarrays, variability of differential expression, low specificity of cDNA microarray probes, discrepancy in fold-change calculation and lack of probe specificity for different isoforms of a gene. CONCLUSIONS: In view of these pitfalls, data from microarray analysis need to be interpreted cautiously.

Alleles↗