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Biomedical subjects

Sanjay Tyagi

Publications and source records attributed to Sanjay Tyagi.

At least 19 recordsLinked to original sources
PubMed2006

Phosphodiesterase-5 inhibitor in Eisenmenger syndrome: a preliminary observational study.

BACKGROUND: Phosphodiesterase-5 inhibitors produce a significant decrease in pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension. We studied the effects of tadalafil, a phosphodiesterase-5 inhibitor, on short-term hemodynamics, tolerability, and efficacy over a 12-week period in patients of Eisenmenger syndrome having a pulmonary vascular pathology similar to idiopathic pulmonary arterial hypertension. METHODS AND RESULTS: Sixteen symptomatic Eisenmenger syndrome patients (mean age, 25+/-8.9 years) were assessed hemodynamically at baseline and 90 minutes after a single dose of tadalafil (1 mg/kg body weight up to a maximum of 40 mg). The same dose was then continued daily for 12 weeks, and the patients were restudied. There was a significant decrease in mean pulmonary vascular resistance immediately (24.75+/-8.49 to 19.22+/-8.23 Woods units; P<0.005) and at 12 weeks (19.22+/-8.23 to 17.02+/-6.19 Woods units; P=0.03 versus 90 minutes). Thirteen of 16 patients (81.25%) showed a > or = 20% decrease in pulmonary vascular resistance and were defined as responders. The mean systemic oxygen saturation improved significantly both immediately (84.34+/-5.47% to 87.39+/-4.34%; P<0.005) and at 12 weeks (87.39+/-4.34% to 89.16+/-3.8%; P<0.02 versus 90 minutes) without a significant change in systemic vascular resistance. None of the patients had a fall in systemic arterial pressure, worsening of systemic oxygen saturation, or any adverse reactions to the drug. The mean World Health Organization functional class improved from 2.31+/-0.47 to 1.25+/-0.44 (P<0.0001), and the 6-minute walk distance improved from 344.56+/-119.06 to 387.56+/-117.18 m (P<0.001). CONCLUSIONS: Preliminary evaluation of tadalafil has shown efficacy and safety in selected patients with Eisenmenger syndrome, warranting further investigation in this subgroup of patients.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2006

Kaposi's Sarcoma-associated herpesvirus lytic switch protein stimulates DNA binding of RBP-Jk/CSL to activate the Notch pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.

Alternative Splicing↗
PubMed2006

Stochastic mRNA synthesis in mammalian cells.

Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise.

Animals↗
PubMed2005

Mechanism of mRNA transport in the nucleus.

The mechanism of transport of mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject of many studies. Using molecular beacons to track single mRNA molecules in living cells, we have characterized the diffusion of mRNP complexes in the nucleus. The mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. The diffusion of mRNP complexes is restricted to the extranucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the complexes to resume their motion after they become stalled. This finding provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process.

Active Transport, Cell Nucleus↗
PubMed2005

Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

BACKGROUND: A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. METHODS: The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. CONCLUSIONS: The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

Animals↗
PubMed2005

tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells.

When oligonucleotide probes are microinjected into cells to image the distribution of RNAs, they are rapidly sequestered into the nucleus. As a result, it is difficult to detect mRNAs in the cytoplasm of living cells. We were able to overcome this process by attaching tRNA transcripts to the probes. We show that when fluorescently labeled tRNAs, tRNAs with extensions at their 5' end, or chimeric molecules in which a molecular beacon possessing a 2'-O-methylribonucleotide backbone is linked to a tRNA, are injected into the nucleus of HeLa cells, they are exported into the cytoplasm. When these constructs are introduced into the cytoplasm, they remain cytoplasmic. These constructs allow the distribution of both the general mRNA population and specific mRNAs to be imaged in living cells. This strategy should also be useful for enhancing the efficacy of antisense oligonucleotides by keeping them in the cytoplasm. Our observations show that the fidelity of the tRNA export system is relaxed for unnatural tRNA variants when they are introduced into the nucleus in large amounts.

Active Transport, Cell Nucleus↗
PubMed2005

Mitral valvuloplasty by inoue balloon under transthoracic echocardiographic guidance.

BACKGROUND: Mitral valvuloplasty traditionally performed under fluoroscopic guidance has emerged as an effective nonsurgical technique for the treatment of symptomatic patients with mitral stenosis. We undertook a study to evaluate the feasibility of performing valvuloplasty by the Inoue balloon solely under transthoracic echocardiographic (TTE) guidance with the backup of transesophaeal echocardiography (TEE) and fluoroscopy. METHODS: Between September 2003 and July 2004, mitral valvuloplasty using the Inoue balloon was performed solely under TTE guidance in the catheterization laboratory with backup of fluoroscopy and TEE in 75 patients (52 female patients, including 18 who were pregnant, and 23 male patients) with symptomatic mitral stenosis (New York Heart Association class II-IV) with satisfactory TTE window and valve morphology suitable for valvuloplasty. RESULTS: The procedure was technically successful (no requirement of TEE or fluoroscopy) in 68 patients (89%). TEE was required for septal puncture in 3 patients whereas fluoroscopic assistance was required to complete the procedure in 4 patients. Procedural success after valvuloplasty (doubling of mitral valve area or mean gradient across mitral valve < 5 mm Hg in absence of complications like severe mitral regurgitation or tamponade) was achieved in 70 patients. There was an increase in mean mitral valve area as assessed echocardiographically from 0.84 +/- 0.16 cm2 to 1.7 +/- 0.27 cm2 with decrease in mean diastolic gradient from 27.2 +/- 8.4 mm Hg (18-36 mm Hg) to 5.2 +/- 4.1 mm Hg (3-14 mm Hg). The mean procedure time was 27.2 +/- 8.4 minutes (15-45 minutes). Mild mitral regurgitation appeared or increased in severity to moderate degree as assessed by color Doppler during the procedure in 11 patients (14.6%) but no patient developed severe mitral regurgitation. No patient had cardiac tamponade as puncture of the septum under TTE guidance prevents inadvertent puncture of the aorta, coronary sinus, inferior vena cava, and right atrial and left atrial wall that sometimes occurs during septal puncture under fluoroscopic guidance. CONCLUSION: Balloon mitral valvuloplasty under sole TTE guidance is safe and feasible in experienced hands.

Adolescent↗
PubMed2005

Nonsurgical management of cardiac tamponade caused by a temporary pacemaker lead.

Right ventricular perforation secondary to temporary pacemaker lead, resulting in cardiac tamponade, is a rare but life-threatening complication. Surgery is usually the definitive management described for such cases. We describe successful sealing of such a perforation by percutaneous instillation of cyanoacrylate glue, through a guiding catheter in the pericardial sac at the perforation site.

Aged↗
PubMed2004

Imaging native beta-actin mRNA in motile fibroblasts.

Nuclease-resistant, cytoplasmically resident molecular beacons were used to specifically label beta-actin mRNA in living and motile chicken embryonic fibroblasts. beta-actin mRNA signals were most abundant in active lamellipodia, which are protrusions that cells extend to adhere to surfaces. Time-lapse images show that the immediate sources of beta-actin mRNA for nascent lamellipodia are adjacent older protrusions. During the development of this method, we observed that conventional molecular beacons are rapidly sequestered in cell nuclei, leaving little time for them to find and bind to their cytoplasmic mRNA targets. By linking molecular beacons to a protein that tends to stay within the cytoplasm, nuclear sequestration was prevented, enabling cytoplasmic mRNAs to be detected and imaged. Probing beta-actin mRNA with these cytoplasmically resident molecular beacons did not affect the motility of the fibroblasts. Furthermore, mRNAs bound to these probes undergo translation within the cell. The use of cytoplasmically resident molecular beacons will enable further studies of the mechanism of beta-actin mRNA localization, and will be useful for understanding the dynamics of mRNA distribution in other living cells.

Actins↗
PubMed2004

Real-time measurement of in vitro transcription.

We have developed a simple method to measure RNA synthesis in real time. In this technique, transcription reactions are performed in the presence of molecular beacons that possess a 2'-O-methylribonucleotide backbone. These probes become fluorescent as they hybridize to nascent RNA during the course of synthesis. We found that molecular beacons synthesized from natural deoxyribonucleotides were not suitable, because they are copied by RNA polymerases, generating complementary product strands that bind to the molecular beacons, causing a conformational change that results in unwanted fluorescence. However, when the molecular beacons are synthesized from 2'-O-methylribonucleotides, they are not copied and fluorescence is strictly dependent upon transcription of the added template. Utilizing these modified molecular beacons, quantitative comparisons were made of the activity of a variety of RNA polymerases and the effect of an inhibitor of transcription was determined.

Bacteriophage T7↗
PubMed2004

Endovascular stent implantation for severe pulmonary artery stenosis in aortoarteritis (Takayasu's arteritis).

Symptomatic pulmonary artery stenosis is a relatively uncommon manifestation of aortoarteritis. We describe a patient of aortoarteritis with severe proximal right pulmonary artery stenosis who presented with dyspnea on exertion and central cyanosis. The pulmonary artery stenosis was successfully relieved by percutaneous transluminal balloon angioplasty and implantation of a balloon-expandable stent. This resulted in immediate improvement in oxygen saturation, disappearance of cyanosis, relief of dyspnea, and marked improvement in right lung perfusion as demonstrated by pre- and postangioplasty technetium lung ventilation-perfusion scans. The changes in the pulmonary arterial wall morphology were detected precisely by intravascular ultrasound imaging.

Adult↗
PubMed2004

Stent implantation in right-sided patent ductus arteriosus to relieve severe cyanosis in adult patient with pulmonary atresia and ventricular septal defect.

Patients with unrepaired pulmonary atresia and ventricular septal defect may develop stenosis of collaterals or shunts to the pulmonary arteries leading to hypoperfusion of lungs and systemic hypoxemia. A 25-year-old female with pulmonary atresia and ventricular septal defect presented with progressively increasing cyanosis and exercise intolerance. A restrictive right-sided patent ductus arteriosus was identified as the main source of pulmonary blood flow. We report transcatheter implantation of a balloon-expandable stent across the stenosed duct to augment the pulmonary blood flow as a palliative management option. Patient had immediate improvement in arterial oxygen saturation from 66% to 85% with excellent clinical improvement and stable oxygen saturation on 8 months of follow-up.

Adult↗
PubMed2003

Visualizing the distribution and transport of mRNAs in living cells.

We have visualized the movements of native mRNAs in living cells. Using nuclease-resistant molecular beacons, we imaged the transport and localization of oskar mRNA in Drosophila melanogaster oocytes. When the localization pattern was altered by genetic manipulation of the mRNA's 3' untranslated region, or by chemical perturbation of the intracellular tubulin network, the distribution of the fluorescence signals changed accordingly. We tracked the migration of oskar mRNA in real time, from the nurse cells where it is produced to the posterior cortex of the oocyte where it is localized. Our observations reveal the presence of a transient, and heretofore elusive, stage in the transport of oskar mRNA. Direct visualization of specific mRNAs in living cells with molecular beacons will accelerate studies of intracellular RNA trafficking and localization, just as the use of green fluorescent protein has stimulated the study of specific proteins in vivo.

3' Untranslated Regions↗
PubMed2003

Endovascular treatment of aortic rupture during angioplasty for aortic in-stent restenosis in aortoarteritis.

Aortic rupture during balloon angioplasty for in-stent restenosis without attempting to overexpand it is a rarity. We report a case of a young woman with aortoarteritis who had aortic rupture during angioplasty for in-stent restenosis. The balloon used was of the same diameter as the previously implanted stent and was completely within the stent during inflation. The disruption was successfully treated by percutaneous placement of a self-expandable endovascular stent graft.

Adult↗
PubMed2003

Self- and balloon-expandable stent implantation for severe native coarctation of aorta in adults.

BACKGROUND: Balloon angioplasty for native coarctation of the aorta (CoA) in adults, though promising, is sometimes limited by significant residual gradient (>20 mm Hg). Few studies available have reported on use of balloon-expandable stents in such a situation. We evaluated the use of self- and balloon-expandable stents in patients with suboptimal response to balloon angioplasty (BA). METHODS: Twenty-one hypertensive patients (age, 18 to 61 years; mean, 28.6 +/- 11.2 years) with native CoA and in whom results of BA were suboptimal (ie, residual peak systolic gradient [PSG] >20 mm Hg) underwent stent implantation. Balloon-expandable Palmaz stents were implanted in 5 patients (group A) and self-expandable nitinol aortic stents in the remaining 16 patients (group B). RESULTS: In group A, PSG decreased from 62.8 +/- 10.6 (53 to 80) mm Hg to 28.1 +/- 6.3 (22 to 39) mm Hg after BA. Systolic gradient further decreased to 8.3 +/- 3.9 (2 to 16) mm Hg (P <.001) after implantation of the balloon-expandable Palmaz stent. In group B, PSG decreased from 70.2 +/- 24.6 (40 to 110) mm Hg to 28.4 +/- 9.8 (22 to 42) mm Hg after BA and further reduced to 9.0 +/- 5.5 (4 to 16) mm Hg (P <.001). One of these patients had a nitinol self-expandable stent implanted after a Palmaz stent embolized immediately after deployment. Nitinol stents were easier to deploy and conformed better to aortic anatomy compared with balloon-expandable stents. In group A, the diameter of the coarcted segment increased from 3.8 +/- 0.8 mm to 13.3 +/- 0.8 mm (P <.001) after stent implantation and in group B it increased from 4.5 +/- 1.1 mm to 14.1 +/- 2.1 mm (P <.001). There was no significant difference between the two groups in the PSG and diameter of the coarcted segment before and after stent implantation. With the exception of one case, in which a Palmaz stent embolized, there was no other complication in our series. On follow-up of 12 to 71 months (mean, 40.7 +/- 5.8 months) all the implanted stents remained in their original position and none showed evidence of fracture. Improvement in hypertension was seen in 20 of 21(95.2%) of the patients. On recatheterization and angiography 1.2 +/- 0.6 years after implantation in 19 patients, one patient showed an increase in PSG to 27 mm Hg across the nitinol stent and underwent successful redilation. No increase in gradient was seen in other patients. Beneficial late remodeling was seen in 10 of 14(71.4%) of patients restudied after implantation of self-expandable stent. None of the patients showed aneurysm formation. CONCLUSIONS: Stent implantation is safe and effective in improving suboptimal results after BA for CoA. Self-expandable stents were easier to implant, adapted better to the wall of the aorta, and in most patients had similar efficacy in reducing coarctation as balloon-expandable stents.

Adult↗
PubMed2003

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR.

We have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis, using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granuloma-specific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis, as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo. The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.

Animals↗