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Biomedical subjects

R Ramalingam

Publications and source records attributed to R Ramalingam.

14 recordsLinked to original sources
PubMed2001

Upregulation of transcription factors in lung in the early phase of postpneumonectomy lung growth.

In the adult rodent, pneumonectomy results in compensatory lung growth characterized by cell proliferation. The molecular mechanisms governing this response remain unknown. We hypothesized that, in the early period postpneumonectomy, upregulated expression of transcription factors drives the growth process. We utilized a cDNA expression array to screen for upregulated transcription factors after left pneumonectomy in adult C57BL/6 mice, using unoperated mice as controls. Quantification of mRNA expression in the remaining lung at 2 h demonstrated a twofold or greater upregulation of six transcription factors: early growth response gene-1 (Egr-1), Nurr77, tristetraprolin, the primary inhibitor of nuclear factor-kappa B (I kappa B-alpha), gut-enriched Krüppel-like factor (GKLF), and LRG-21. Northern analysis was used to quantify the upregulation of expression of these genes relative to sham thoracotomy and unoperated controls. The largest increase was in Egr-1 (4.7-fold > naive). Time-course analysis over the first 24 h confirmed the transient nature of the early upregulation. In the context that postpneumonectomy lung growth is associated with cell proliferation and that genes such as Egr-1, Nurr77, LRG-21, and tristetraprolin have known roles in stress response, vascular biology, embryology, and cellular development, these data support the concept that transcription factors function early in the cascade of events leading to the compensatory response.

Animals↗
PubMed2000

Downregulation of CXCR4 gene expression in primary human endothelial cells following infection with E1(-)E4(+) adenovirus gene transfer vectors.

Infection of human endothelial cells with first-generation E1(-)E4(+) adenovirus (Ad) vectors leads to prolonged cell survival and changes in the cell phenotype to a more quiescent stage. Based on the concept that the CXCR4, the receptor for the endothelial chemoattractant stromal-derived factor-&alpha (SDF-alpha), is constitutively expressed by quiescent, resting endothelial cells, the present study analyzes the effect of Ad vector infection on CXCR4 expression and SDF-alpha responses of human umbilical vein endothelial cells (HUVEC). CXCR4 transcripts were markedly downregulated in E1(-)E4(+) Ad-infected cells 48 h following infection, but not in uninfected control cells or when the cells were infected with an E1(-)E4(-) Ad vector. Analysis of surface CXCR4 expression by flow cytometry demonstrated marked reduction of the CXCR4 receptor on cells infected with E1(-)E4(+) Ad compared to uninfected control cells or E1(-)E4(-) Ad-infected cells. Infection of other cell types which express CXCR4, such as dendritic cells and myeloma cells, did not exhibit CXCR4 receptor downregulation following infection with E1(-)E4(+) Ad. Consistent with the observed downregulation of CXCR4 mRNA and surface protein, infection of the endothelial cells with an E1(-)E4(+) Ad rendered the cells unresponsive to the chemoattractant SDF-alpha compared to naive or E1(-)E4(-) Ad-infected cells. Together, the data suggest that first-generation Ad vectors, likely the E4 region, modify the ability of endothelial cells to respond to at least one important chemoattractant.

Adenoviridae↗
PubMed1999

E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells.

Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1(-)E4(+) replication-deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1(-)E4(+) Ad vectors provide an "antiapoptotic" signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of "suspended animation," remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a "pro-life" program that modifies cultured endothelial cells to survive for prolonged periods.

Adenoviridae↗
PubMed1999

Induction of endogenous genes following infection of human endothelial cells with an E1(-) E4(+) adenovirus gene transfer vector.

Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1(-) E4(+) Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein alpha type S [Galpha-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor beta2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1(-) E3(-) Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.

Adenovirus E1 Proteins↗
PubMed1999

Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor.

Robust expression of genes transferred by adenovirus (Ad) vectors depends upon efficient entry of vectors into target cells. Cells deficient in the coxsackie/adenovirus receptor (CAR) are difficult targets for Ad-mediated gene transfer. We hypothesized that low levels of CAR expression may be responsible, in part, for the relative inefficiency of Ad-mediated gene transfer to human alveolar macrophages (AMs). CAR gene expression was detected in human AMs by reverse transcription-polymerase chain reaction and at low levels by Northern analysis. Indirect immunofluorescence showed specific, low-intensity surface staining for CAR, but at levels below those found on the positive-control A549 human lung epithelial cell line. Consistent with this, AMs expressed Ad vector transgenes 100 to 1,000-fold less efficiently than A549 cells, as assessed using the beta-galactosidase reporter (chemiluminescence assay) and green fluorescent protein (fluorescence microscopy and flow cytometry). At high multiplicity of infection, AMs from an HIV+ individual could be transduced with an AdIFNgamma vector to secrete detectable human interferon-gamma. Ad transgene expression by AMs was blocked by capsid fiber protein, suggesting that CAR is required in the pathway for productive Ad entry into alveolar macrophages. To confirm that Ad transgene expression by AMs is limited by low levels of CAR expression, cells were infected with an Ad vector containing the CAR complementary DNA (cDNA). Enhanced expression of CAR protein was demonstrated by indirect immunofluorescence, and the CAR cDNA-transduced cells showed 5-fold enhancement of subsequent Ad transgene expression. These observations demonstrate that human AMs can be targets for Ad-mediated gene transfer, but that efficiency of transgene expression is limited, at least in part, by low levels of CAR expression.

Adenoviridae↗
PubMed1999

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus.

We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.

Adenoviridae↗
PubMed1997

Characterization of the Dictyostelium discoideum cellulose-binding protein CelB and regulation of gene expression.

Similar to other stages of Dictyostelium development, spore germination is a particularly suitable model for studying regulation of gene expression. The transition from spore to amoeba is accompanied by developmentally regulated changes in both protein and mRNA synthesis. A number of spore germination-specific cDNAs have been isolated previously. Among these are two members of the 270 gene family, a group of four genes defined by the presence of a common tetrapeptide repeat of Thr-Glu-Thr-Pro. celA (formerly called 270-6) and celB (formerly 270-11) are expressed solely and coordinately during spore germination, the levels of the respective mRNAs being low in dormant spores, rising during germination to a maximum level at about 2 h, and then rapidly declining as amoebae are released from spores. The mRNAs are not found in growing cells or during multicellular development. The rapidity with which these transcripts accumulate and then disappear during germination implies that the respective products may be important for the process. We reported previously that the CelA protein is a cellulase (endo-1, 4-beta-glucanase (EC 3.2.1.4)). In the present investigation, properties of the CelB protein, a glycosylated protein of 532 amino acids, 36% of which are serine or threonine, were examined, and the upstream sequences involved in the developmental regulation of the expression of the gene have been determined. The CelB protein does not demonstrate cellulase activity, but it has a cellulose-binding domain. Its role, if any, in degradation of the cellulose-containing spore wall is unknown. To identify cis-acting elements in the celB promoter, unidirectional 5' deletions of the celB upstream noncoding region were constructed and used to transform amoebae. Analysis of promoter activity during different stages of development shows that a short, very A/T-rich sequence of approximately 81 base pairs is sufficient for spore-specific celB transcription. Contained in this sequence is the Myb oncogene protein binding site, TAACTG, which was shown previously to be a negative regulator of celA transcription. Dictyostelium and mouse Myb proteins bind to this region of the promoter, suggesting that Myb might regulate celB gene expression negatively as it does in celA.

Animals↗
PubMed1997

Genetics of otosclerosis.

Otosclerosis is an early-middle adult life genetic disease affecting bone remodelling in the ear. Current knowledge of otosclerosis as an inherited disease dates to the mid-19th century, and we report here an attempt to understand the genetics of otosclerosis and detect its heterogeneity. The analysis was conducted on 151 otosclerotic families. The results of our study indicate that while heredity plays an important role in the manifestation of the disease a substantial portion of otosclerotic cases could arise due to non-genetic causes.

Adolescent↗
PubMed1997

HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B.

The HIV-1 accessory gene product Vpr can influence viral pathogenesis by affecting viral replication as well as host cell transcription and proliferation. We have investigated the effects of Vpr on host cell activation and confirm that it influences cellular proliferation. However, we have also found that Vpr modulates T-cell receptor (TCR)-triggered apoptosis in a manner similar to that of glucocorticoids. In the absence of TCR-mediated activation, Vpr induces apoptosis whereas in its presence, Vpr interrupts the expected induction of apoptosis. This regulation of apoptosis is linked to Vpr suppression of NF-kappa B activity via the induction of I kappa B, an inhibitor of NF-kappa B. Further, Vpr suppresses expression of IL-2, IL-10, IL-12, TNF alpha and IL-4, all of which are NF-kappa B-dependent. The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr.

Animals↗
PubMed1995

AT-rich upstream sequence elements regulate spore germination-specific expression of the Dictyostelium discoideum celA gene.

Two members of a family of spore germination-specific cDNAs, celA and celB, are expressed coordinately, exclusively during spore germination. In the present study the regulatory sequence elements responsible for celA germination-specific expression have been identified. The very AT-rich 81 bp sequence between -664 and -584 upstream of the translation initiation site was required for proper temporal transcription of the celA gene. This sequence is comprised of two cis elements, each of which was active by itself in allowing celA expression. Electrophoretic mobility shift assays showed that a factor(s) in an extract prepared from germinating spores bound to the celA regulatory region. One of the three complexes formed was specific for the germinating spore extract. The results are consistent with the notion that the factor(s) that binds to this regulatory region is involved in expression of celA.

Animals↗
PubMed1993

Cloning and functional expression of a Dictyostelium discoideum protein tyrosine phosphatase.

Using polymerase chain reaction methods, we cloned a 1.7-kilobase cDNA, denoted DdPTPa, that has high homology with other known eukaryotic protein tyrosine phosphatases. DdPTPa possess a 241-amino acid protein tyrosine phosphatase domain located in the C terminus, which exhibits a 39-43% amino acid sequence identity with published protein tyrosine phosphatases. Absence of a characteristic signal sequence and transmembrane domain suggests that DdPTPa is a nonreceptor type cytoplasmic protein tyrosine phosphatase. Southern blot analysis of genomic DNA indicates the presence of a multigene protein tyrosine phosphatase family in Dictyostelium. Northern blot analysis reveals four species of mRNA that hybridize to the DdPTPa probe, at least three of which are developmentally regulated. The entire coding sequence of DdPTPa was subcloned into the pET15-b vector and expressed in Escherichia coli. Affinity-purified DdPTPa protein efficiently dephosphorylates both p-nitrophenyl phosphate and tyrosine-phosphorylated reduced, carboxyamidomethylated, and maleylated lysozyme. A Dictyostelium transformant overexpressing DdPTPa does not develop normally. The overexpresser fails to aggregate, in contrast to the control transformant containing vector alone, and after 24 h gives rise to only a few abnormal slugs and small fruiting bodies.

Amino Acid Sequence↗
PubMed1992

The Dictyostelium discoideum spore germination-specific cellulase is organized into functional domains.

During Dictyostelium discoideum spore germination, degradation of the cellulose-containing spore wall is required to allow the amoeba to emerge. The CelA gene, which is transcribed and expressed exclusively during spore germination, codes for a 705-amino-acid protein that has cellulase activity [endo-(1,4)-beta-D-glucanase]. Amoebae transformed by a vector containing the CelA coding sequence or portions of it transcribed from a heterologous promoter expressed and secreted full-length or suitably truncated proteins during vegetative growth when, under normal conditions, these proteins are not made. The gene constructs divided the CelA protein into three domains: a 461-amino-acid N-terminal region that has significant similarity to those of other cellulases and that has been shown to be the catalytic domain; a contiguous 91-residue repeat containing the motif threonine-glutamic acid-threonine-proline, which is glycosylated; and, joined to the repeat, a C-terminal 153-amino-acid sequence that most probably defines a cellulose-binding domain.

Animals↗