Biomedical subjects

S Roy

Publications and source records attributed to S Roy.

At least 235 records · Page 13Linked to original sources

PubMed1999

even-skipped determines the dorsal growth of motor axons in Drosophila.

Axon pathfinding and target choice are governed by cell type-specific responses to external cues. Here, we show that in the Drosophila embryo, motorneurons with targets in the dorsal muscle field express the homeobox gene even-skipped and that this expression is necessary and sufficient to direct motor axons into the dorsal muscle field. Previously, it was shown that motorneurons projecting to ventral targets express the LIM homeobox gene islet, which is sufficient to direct axons to the ventral muscle field. Thus, even-skipped complements the function of islet, and together these two genes constitute a bimodal switch regulating axonal growth and directing motor axons to ventral or to dorsal regions of the muscle field.

Animals↗

PubMed1999

Selective venous thrombolysis with the nipple-balloon catheter: comparative evaluation in vivo.

PURPOSE: To compare in an animal model of deep vein thrombosis, an intramural drug delivery catheter, the nipple-balloon catheter, with an occlusion balloon-infusion guide wire system. MATERIALS AND METHODS: Ten juvenile pigs were used for the study. Deep vein thrombosis was induced in both hind limbs by using a previously described technique. Heparin was administered 30 minutes later (2,500 IU intravenously) and bilateral thrombolysis was attempted with use of 8 mg of alteplase as a 0.25 mg/mL solution containing heparin 50 IU/mL (n = 10) and sodium/meglumine ioxaglate 40 mgI2/mL (n = 5). In one limb, the external iliac vein was endoluminally occluded, and 0.8 mL of alteplase was administered every 3 minutes through a multisideport infusion wire placed coaxially through the balloon catheter. On the other side, a nipple-balloon catheter was used: alteplase was injected as two 0.4-mL aliquots every 3 minutes in overlapping segments of the vessel. Blood samples were taken at predetermined intervals to determine the partial thromboplastin time and plasma fibrinogen concentration. At autopsy, the thrombus mass in the iliofemoral veins was measured, and the extent of residual thrombosis in the venous tributaries was graded at four sites. The heart and the lungs were also examined for thromboemboli (n = 5). Venous specimens were then subjected to X-ray fluorescence spectrometry to determine iodine content (n = 5). RESULTS: Bilateral thrombolysis could be successfully completed in all animals. No procedural problem associated with the use of the nipple-balloon catheter was encountered. The mass of residual thrombus in the axial veins was significantly lower in this group (P = .005). The drug delivery system used did not appreciably influence thrombolysis in the tributaries. Signs of macroscopic damage to the veins were not observed in any animal. None of the venous specimens had detectable levels of iodine. Small thromboemboli were found in the pulmonary circulation in three of five animals. Fibrinogen levels did not decrease during the procedure. CONCLUSIONS: The significantly lower residual thrombus burden associated with use of the nipple-balloon catheter suggests that the device may have the potential to be an effective delivery system for selective thrombolysis in veins.

Animals↗

PubMed1999

In vivo evaluation of the adjustable temporary venous spring filter and the RF02 temporary filter: comparative study.

RATIONALE AND OBJECTIVES: The authors performed this study to compare the in vivo efficacies of the temporary venous spring filter and the RF02 filter in an animal model. MATERIALS AND METHODS: Either the spring filter or the RF02 filter was placed in the inferior vena cava of 10 pigs each, and two clots (5 x 20 mm) were funneled into the filters at 1-hour intervals. The second clots were funneled without removing the first clots captured by the filters. Clot-trapping ability, caval occlusion associated with the clot-trapping procedure, arterial blood gas concentrations, and changes in arterial and iliac venous pressures were evaluated. RESULTS: Placement of the RF02 filter caused elevation of iliac venous pressure with a maximum of 2.2 mm Hg (median) (n = 13, P = .003). Placement of the spring filter parallel to venous flow enabled capture of 90% (nine of 10) and 100% (six of six) of the first and second clots, respectively. The RF02 filter captured clots consistently. The difference between filters was not statistically significant. Both filters equally contributed to elevation of iliac venous pressure (median, 9.3 and 7.2 mm Hg [n = 9] with the spring filter and RF02 filter, respectively). Caval occlusion occurred in 17% (one of six) and 67% (six of nine) of animals after two clots were trapped in the spring filter and RF02 filter, respectively (P = .06). Other parameters were not influenced by the clot-trapping procedure. CONCLUSION: Although a larger version should be developed and better stability of the filter is needed, the spring filter proved to be an efficient filtering device and had a lower rate of caval occlusion compared with the RF02 filter.

Animals↗

PubMed1999

Transcatheter aspiration: the key to successful percutaneous treatment of deep venous thrombosis?

RATIONALE AND OBJECTIVES: The purpose of this study was to examine transcatheter aspiration as an adjunct to local thrombolysis in a porcine model of acute deep venous thrombosis (DVT). MATERIALS AND METHODS: DVT was induced in both hind limbs of five pigs. Thirty minutes later, bilateral thrombolysis was performed by using infusion guidewires placed coaxially through occlusion balloon catheters. A temporary venous filter was then placed in the inferior vena cava. The balloon catheters and infusion wire were removed, a 14-F sheath was placed on the right side, and aspiration of residual thrombi was attempted with 8- and 6-F catheters. At autopsy, the mass of any thrombus in the iliofemoral veins was measured, and residual thrombosis in the venous tributaries was graded at four sites. Thromboemboli in the inferior vena cava and the pulmonary circulation were also collected and weighed. RESULTS: With aspiration and lysis, the iliofemoral veins were cleared of thrombus in three of five limbs; from the remaining two, only 0.09 and 0.15 g of thrombus were harvested. Except for the deep femoral vein in two legs, tributaries were free of thrombus. After thrombolysis alone, thrombi ranged in mass from 0.54 to 1.14 g (median, 0.70 g). Some thrombi were observed in most tributaries. One or two small emboli were found trapped by the caval filter and in the pulmonary circulation in four and three pigs, respectively. CONCLUSION: The primary axial veins and their tributaries can be rapidly cleared of thrombus by using thrombolysis with transcatheter aspiration, but this procedure is associated with the risk of pulmonary embolism.

Acute Disease↗

PubMed1999

Dominant-negative caveolin inhibits H-Ras function by disrupting cholesterol-rich plasma membrane domains.

The plasma membrane pits known as caveolae have been implicated both in cholesterol homeostasis and in signal transduction. CavDGV and CavKSY, two dominant-negative amino-terminal truncation mutants of caveolin, the major structural protein of caveolae, significantly inhibited caveola-mediated SV40 infection, and were assayed for effects on Ras function. We find that CavDGV completely blocked Raf activation mediated by H-Ras, but not that mediated by K-Ras. Strikingly, the inhibitory effect of CavDGV on H-Ras signalling was completely reversed by replenishing cell membranes with cholesterol and was mimicked by cyclodextrin treatment, which depletes membrane cholesterol. These results provide a crucial link between the cholesterol-trafficking role of caveolin and its postulated role in signal transduction through cholesterol-rich surface domains. They also provide direct evidence that H-Ras and K-Ras, which are targeted to the plasma membrane by different carboxy-terminal anchors, operate in functionally distinct microdomains of the plasma membrane.

3T3 Cells↗

PubMed1999

Reduction of fibronectin expression by intravitreal administration of antisense oligonucleotides.

We have investigated whether antisense oligonucleotides delivered intravitreally could reduce gene expression specifically in the retina. In this study, phosphorothioate antisense oligonucleotides targeted to fibronectin transcripts were coupled to a novel carrier and used to specifically reduce fibronectin (FN) expression in retinal vascular cells. Using confocal microscopy, fluorescence from fluorescein isothio-cyanate-labeled FN-oligonucleotides was detected in retinal vascular cells at 24 h postinjection and persisted until day 6 (the end point of this study). The fibronectin mRNA level was consistently decreased to 86.7% +/- 7.9% of control (p<0.05) at day 2, and 46.7% +/- 4.9% of control (p<0.01) at day 6. In contrast, the beta-actin mRNA level, an internal control, was unaltered in rat retinas that received FN-oligonucleotides. Fibronectin protein level at day 6 was also significantly reduced to 61.4% +/- 16% of control (p<0.01). No toxic effect resulting from the carrier was detected histologically. Thus, intravitreal delivery of antisense oligonucleotides to modulate abnormal gene expression in retinal diseases may be an effective approach for ocular gene therapy.

Animals↗

PubMed1999

In vivo evaluation of a novel epitope-tagged human factor VIII-encoding adenoviral vector.

Haemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and is an attractive target for gene therapy. Adenoviral vectors encoding a human B-domain deleted (BDD) FVIII cDNA have been shown previously to mediate expression of high levels of human FVIII and correct the bleeding defect in haemophiliac mice and dogs. While vector assessment in a non-human primate model would have a significant preclinical benefit, a haemophiliac non-human primate model is not available, and assays that distinguish human FVIII from monkey FVIII have not been developed successfully. As a first step to enable vector evaluation in non-human primates, we have constructed an epitope-tagged FVIII molecule by the addition of 16 amino-acids to the carboxy terminus of the BDD protein (BDD-E). Following vector administration to normal mice, therapeutic levels of BDD-E FVIII were expressed for at least 20 weeks. Treatment of haemophiliac mice revealed that the BDD-E protein was biologically active in vivo. To distinguish the BDD-E protein from non-human primate FVIII, a sensitive immunoprecipitation/Western assay was developed that reproducibly detected 1 ng mL-1 of the epitope-tagged human FVIII in the presence of monkey plasma. These data demonstrate that the addition of an epitope tag had no effect on FVIII function or immunogenicity, and suggest that the BDD-E vector will be an effective reagent for non-human primate studies.

Adenoviridae↗

PubMed1999

Linear free-energy model description of the conformational stability of uracil-DNA glycosylase inhibitor A thermodynamic characterization of interaction with denaturant and cold denaturation.

The equilibrium unfolding of uracil DNA glycosylase inhibitor (Ugi), a small acidic protein of molecular mass 9474 Da, has been studied by a combination of thermal-induced and guanidine hydrochloride (GdnCl)-induced denaturation. The analysis of the denaturation data provides a measure of the changes in conformational free energy, enthalpy, entropy and heat capacity DeltaCp that accompany the equilibrium unfolding of Ugi over a wide range of temperature and GdnCl concentration. The unfolding of Ugi is a simple two-state, reversible process. The protein undergoes both low-temperature and high-temperature unfolding even in the absence of GdnCl but more so in the presence of denaturant. The data are consistent with the linear free-energy model and with a temperature independent DeltaCp over the large temperature range of unfolding. The small DeltaCp (6.52 kJ.mol-1.K-1) for the unfolding of Ugi, is perhaps a reflection of a relatively small, buried hydrophobic core in the folded form of this small monomeric protein. Despite a relatively low value of DeltaG(H2O), 7.40 kJ.mol-1 at pH 8.3, Ugi displays considerable stability with the temperature of maximum stability being 301.6 K.

Cold Temperature↗

PubMed1999

Methods to assess free radicals and oxidative stress in biological systems.

Oxidative stress results from a disruption of the prooxidant/antioxidant cellular balance and monitoring free radical status becomes an interesting challenge in animal and human nutrition. In the present work, merits and limitations of different analytical techniques (HPLC, GC-MS, fluorometric and colourometric assays, ELISA, gel electrophoresis) for the measurement of radical mediated alterations in the cellular integrity of lipids (malondialdehyde, hydrocarbon gases, F2-isoprostanes) proteins (protein carbonyls, 3-nitrotyrosine) and DNA (8-hydroxy-2'-deoxyguanosine) are discussed. Besides these indirect methods, owing to the fact that free radicals are paramagnetic, electron paramagnetic resonance spectroscopy combined with spin trapping has become a valuable tool to directly assess and to better understand the mechanisms of free radical reactions. With this approach a radical that is too short-lived to be detected, adds to a spin-trapping agent to form a relatively long-lived radical adduct. Information obtained from the hyperfine splitting of the spin-trapped adduct can provide identification and quantification of the originally generated free radicals.

Animals↗

PubMed1999

Solid-state phase transitions of AG337, an antitumor agent.

The object of this investigation was to perform detailed solid-state characterization studies on the different solid forms of AG337 and to determine the conditions of their interconversions. Solid-state characterization was done using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), hot stage microscopy, Karl Fischer titrimetry, ambient and variable temperature X-ray powder diffractometry (XRD) and TGA coupled with FTIR (TGA/FTIR). In addition to five polymorphic forms of the anhydrate (I alpha to I epsilon), a hemihydrate (C14H12N4OS.2HCl.0.5H2O, II), a monohydrate (C14H12N4OS.2HCl.H2O; III), as well as a dihydrate (C14H12N4OS.2HCl.2H2O; IV) were identified. The 'as is' anhydrate, I alpha, resisted water uptake until stored at 98% RH (room temperature), where it transformed directly to IV, II and III transformed to IV at RH values > or = 7.6 and 84% respectively. Heating II and III to 130 degrees C in the variable temperature XRD resulted in the formation of I beta and I gamma respectively. On the other hand, I delta and I epsilon were obtained when II and III were respectively stored at 60 degrees C under vacuum. Variable temperature XRD, by providing information about the solid-state as a function of temperature, assisted in the interpretation of the DSC and TGA results. TGA/FTIR provided direct evidence that the thermal events observed in the temperature ranges of 25-150 degrees C and 200-250 degrees C were due to loss of water and loss of hydrogen chloride respectively. In addition to the conventional analytical techniques such as XRD, DSC, TGA and KFT, two other techniques, (variable temperature XRD and TGA/FTIR), were very useful in these solid-state characterization studies.

Antimetabolites, Antineoplastic↗

PubMed1999

Association of vitamin D receptor genotype with leprosy type.

Host genetic factors including major histocompatibility complex (MHC) polymorphisms influence both susceptibility to leprosy per se and also to leprosy type. Non-MHC genes may play an important role, but such genes remain undefined. The influence of two non-MHC candidate genes was assessed in a case-control study of Bengali leprosy patients from Calcutta. Recent studies have implicated variation in the vitamin D receptor (VDR) gene in susceptibility to several diseases, including osteoporosis and pulmonary tuberculosis. In this population, homozygotes for the alternate alleles of the VDR polymorphism are associated, respectively, with lepromatous and tuberculoid leprosy. The NRAMP1 (natural resistance associated macrophage protein 1) gene may influence human mycobacterial disease susceptibility based on studies with the murine homologue Nramp1. However, no significant association was found between NRAMP1 and leprosy susceptibility. This study suggests that the VDR polymorphism may influence susceptibility to some diseases by affecting the type and the strength of the host immune response.

Alleles↗

PubMed1999

High expression of human 15-lipoxygenase induces NF-kappaB-mediated expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and T-cell adhesion on human endothelial cells.

Expression of 15-lipoxygenase (15-LO) is induced over 100-fold in early fatty streak lesions. 15-LO activity leads to the production of specific lipid hydroperoxides, which can have major effects on the expression of proinflammatory genes involved in atherogenesis. We have used retrovirus-mediated gene transfer to achieve stable high expression of 15-LO in human endothelial ECV304 cells. These cells were used to study the effects of 15-LO on the expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), activation of nuclear factor kappa B (NF-kappaB), and T-cell adhesion on endothelial cells. NF-kappaB activation was greatly potentiated by increased 15-LO activity in the stably transduced cells, and both VCAM-1 and ICAM-1 were significantly induced in these cells in response to tumor necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA) stimulation, as studied by flow cytometry. The induction of ICAM-1 was sensitive to antioxidants in a dose-dependent manner. The adherence of Jurkat T cells on the 15-LO-expressing endothelial cells was markedly induced after PMA stimulation. These results indicate that 15-LO activity may be involved in the early pathogenesis of atherosclerosis by inducing VCAM-1 and ICAM-1 expression and by increasing T-cell adhesion on the endothelium.

Antioxidants↗

PubMed1999

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor.

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.

Amino Acid Substitution↗

PubMed1999

Automatic detection of left ventricular contours from cardiac cine magnetic resonance imaging using fuzzy logic.

RATIONALE AND OBJECTIVES: Gated cardiac cine magnetic resonance imaging provides accurate dynamic data of the left ventricular function. However, the manual extraction of important physiologic parameters such as myocardium wall thickness and left ventricular volumes is invariably time consuming and subjective. To reduce the variability and time constraints inherent in observer contour tracing, the authors developed an automatic left ventricle contour-detection method. METHODS: The purpose was to apply fuzzy logic-based automatic contour detection to identification of endocardial and epicardial borders in short-axis magnetic resonance images. The automatic contouring was compared with manual tracing using the calculated ejection fraction as the comparison criterion. RESULTS: A good correlation was found between the two approaches (r2 = 0.98). CONCLUSIONS: The ejection fraction can be obtained using this automatic contouring method.

Adult↗

PubMed1999

Needle cricothyroidotomy revisited.

Needle cricothyroidotomy may provide a life-saving airway when tracheal intubation is not possible. Indications for needle cricothyroidotomy are discussed. Methods of needle/angiocatheter insertion and proposed means to connect to an oxygen source for intermittent insufflation are reviewed. A new technique for administering effective oxygenation and ventilation through a trans-tracheal catheter using materials commonly available in an emergency department is presented. Potential complications are discussed. Complete upper airway obstruction is a contraindication to needle cricothyroidotomy because of the risks of barotrauma. In a crisis situation, the emergency practitioner needs a simple, reliable, effective, and preplanned technique to deal with the "nightmare airway."

Airway Obstruction↗

PubMed1999

A gene encoding a hevein-like protein from elderberry fruits is homologous to PR-4 and class V chitinase genes.

We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene.

Amino Acid Sequence↗

PubMed1999

Generation of an adenovirus vector lacking E1, e2a, E3, and all of E4 except open reading frame 3.

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.

Adenovirus E1 Proteins↗

PubMed1999

MR imaging of the heart in patients after myocardial infarction: effect of increasing intersection gap on measurements of left ventricular volume, ejection fraction, and wall thickness.

PURPOSE: To determine the extent to which the number of planes imaged at magnetic resonance (MR) imaging could be reduced without modifying the calculated volume and thickness of the left ventricle. MATERIALS AND METHODS: Sixty-one patients were examined after a myocardial infarction. The whole left ventricle was imaged by using 5-mm contiguous breath-hold cine MR short-axis sections with no gap (SAng) (two-dimensional fast low-angle shot sequence, 9/4.8 [repetition time msec/echo time msec]). The effect of omitting one section in two (short-axis sections with 5-mm gap [SA5 mm]) or two sections in three (short-axis sections with 10-mm gap [SA10 mm]) was studied. RESULTS: In the comparison of SA5 mm or SA10 mm with respect to the reference SAng, the standard error of the estimate (SEE) for the diastolic volume did not exceed the 6.1% interobserver SEE, and the SEE for the ejection fraction remained lower than the 3% interobserver SEE. The measured wall thickness was not affected. In addition, six simple geometric models were compared with SAng and yielded an SEE of 9.5%-28.1% for the diastolic volume and 3.8%-13.3% for the ejection fraction. CONCLUSION: In the study of left ventricles with heterogeneous contractility, short-axis imaging is more accurate than geometric modeling and permits wall thickness measurements when an intersection gap of 5 or 10 mm is used.

Adult↗