Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complexing recombinant DNA with cationic liposomes is a convenient means of introducing foreign genes into cells (lipofection) and could potentially form the basis for genetically modifying diseased blood vessels in patients. The mechanism of lipofection is incompletely understood but it is recognized that the degree of successful gene transfer is highly dependent on cell type. We have transfected primary cultures of human vascular smooth muscle cells with a plasmid expressing either firefly luciferase (Luc) or nuclear-localized beta-galactosidase (NL-beta-gal). Cells were derived from either normal human internal mammary arteries, fragments of primary atherosclerotic plaque, or fragments of restenotic lesion. Concurrent lipofection of rabbit vascular smooth muscle cells and NIH 3T3 cells was performed as well. Compared to NIH 3T3 cells, expression in human vascular smooth muscle cells was markedly reduced: in cells derived from internal mammary artery, Luc expression, normalized for protein content, was 123-fold lower than in NIH 3T3 cells, while the proportion of cells expressing NL-beta-gal was 30-fold lower. Luc expression in cells derived from restenotic tissue was significantly greater than from cells derived from primary plaque. Within a given population of cells, the mitotic index of cells expressing the recombinant gene was significantly higher than the mitotic index for the total population of cells (p < 0.05). Finally, cotransfection experiments, in which lipofection of smooth muscle cells was performed using genes for NL-beta-gal and for human growth hormone, showed that among positive transfectants, a high proportion of cells (23-36%) co-expressed both genes. Thus, the efficiency of successful lipofection in human vascular smooth muscle cells in vitro is low. Transfection appears to be preferentially facilitated in cells derived from restenotic tissue and specific properties of smooth muscle cells, including growth rates, appear to be critical for successful transfection. Further elucidation of cell properties that promote transfection is required to augment the efficiency of liposome-mediated gene transfer in human vascular cells.
Semin Interv Cardiol 1996 Mar
PMID:Vascular applications of human gene therapy. 955 98

This study was designed to determine if adenoviral-mediated delivery of a transgene encoding the beta 2-adrenergic receptor (beta 2-AR) to the carotid arterial wall could result in alterations in in vivo vascular function. De-endothelialized rat carotid arteries were infused in vivo with 0.1 mg/ml elastase and adenovirus [6 x 10(9) plaque forming units (PFU)] containing either the marker gene beta-galactosidase (Adeno-beta-gal), DNA encoding the human beta 2-AR (Adeno-beta 2-AR), or no transgene. This low concentration of elastase increased the water permeability (5.2 +/- 0.6 v 1.9 +/- 0.4 x 10(-8) cm/s/mmHg, n = 4, P < 0.0001) without affecting either the vasomotor responsiveness or the morphology of the arterial wall. A transfection efficiency of 73% was achieved with Adeno-beta-gal (n = 3). beta-gal expression was associated with infrequent appearance of T and B lymphocytes, or neutrophil infiltration. Five days after infection with Adeno-beta 2-AR, the total beta-AR density increased six-fold (67.8 +/- 3.4 v 397.0 +/- 155.5 fmol/mg protein, n = 5, P < 0.01); isoproterenol-induced vasorelaxation at transmural pressures from 10-110/mmHg increased (P < 0.01) compared to arteries exposed to control virus (empty adenovirus), n = 4; and isoproterenol-stimulated cAMP production was increased by 65% (n = 5). Thus, adenoviral-mediated delivery of beta 2-ARs into large artery walls results in enhanced beta-AR-mediated vasorelaxation via augmentation in cAMP levels in vascular smooth muscle cells.
J Mol Cell Cardiol 1998 May
PMID:Enhanced vasorelaxation by overexpression of beta 2-adrenergic receptors in large arteries. 961 44

It has recently emerged that transcriptional differences exist between left and right cardiac chambers. An example is provided by transgenic mice with an nlacZ reporter gene under transcriptional control of the fast skeletal muscle alkali myosin light chain (MLC) 3 promoter and 3' enhancer, which express beta-galactosidase in a left ventricular-right atrial dominant pattern in the developing and adult heart. Here, we demonstrate that endogenous MLC3F transcripts are also left/right regionalised in the mouse heart during embryonic development. Regionalisation is observed as early as embryonic day (E) 8.5, and by E10.5 MLC3F transcripts are present predominantly in the future left ventricle and right atrium, and to a lesser extent in the left atrium. Subsequently, MLC3F transcripts are down-regulated in the left ventricle, and by E12.5 expression is restricted to both atria and left-ventricular trabeculae. No MLC3F protein can be detected in the adult or embryonic mouse heart, suggesting that post-transcriptional regulation prevents this fast myosin isoform contributing to myocardial contraction. Left ventricular-right atrial dominant MLC3F transgenes therefore reflect transitory left/right regionalisation of the endogenous gene, unlike other reported cases of transgene regionalisation. MLC3F transgenes, however, maintain an embryonic-like distribution throughout development suggesting that myocardial gene expression is controlled by distinct temporal, as well as spatial, regulatory modules.
J Mol Cell Cardiol 1998 Jun
PMID:Dynamic left/right regionalisation of endogenous myosin light chain 3F transcripts in the developing mouse heart. 968 82

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.
J Mol Cell Cardiol 1998 Jul
PMID:Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors. 971 Aug 2

Adenoviral vectors are promising agents for a number of in vivo gene therapy applications including diseases of the heart and coronary vessels. Efficient intravascular gene transfer to specific sites has been achieved in occluded vessels, but otherwise is hampered by the effect of blood flow on localized vector uptake in the vessel wall. An alternative delivery approach to coronary arteries is the expression of diffusible gene products into the pericardial space surrounding the heart and coronary arteries. However, in vivo pericardial access is comparatively difficult and has been limited to surgical approaches. We hypothesized that efficient adenovirus-mediated gene expression in pericardial lining mesothelium could be achieved by transmyocardial vector delivery to the pericardium. To evaluate this concept, a hollow, helical-tipped penetrating catheter was used to deliver vector-containing fluid directly into the intrapericardial space. The catheter was introduced percutaneously in anesthetized mongrel dogs, advanced into the right ventricle, and the tip passed through the apical right ventricular myocardium under direct radiographic visualization until the open end of the catheter tip resided in the intrapericardial space. Adenoviral vectors expressing either nuclear-localizing beta-galactosidase, cytoplasmic luciferase, or secreted human alpha 1AT reporters (Av1nBg, Av1Lu, or Av1Aa, respectively) were instilled through the catheter into the intrapericardial space. Three days later the animals were sacrificed and reporter gene expression was evaluated in pericardium, epicardium, and multiple other tissues. In animals receiving Av1nBg, beta-galactosidase activity was evident in most of the pericardial lining endothelium, up to 100% in many areas. In animals receiving Av1Lu, luciferase reporter activity was abundant in pericardial tissues, but near-background levels were observed in other organs. In animals receiving Av1Aa, human alpha 1AT was abundant (16-29 mg/ml) in pericardial fluid, but was undetectable in serum. All animals tolerated the procedure well with no electrocardiographic changes and no clinical sequelae. These observations demonstrate highly efficient adenovirus vector delivery and gene transfer and expression in the pericardium and support the feasibility of localized gene therapy via catheter-based pericardial approaches. We suggest that the pericardial sac may serve as a sustained-release protein delivery system for the generation of desired gene products or their metabolites for diffusion into the epicardial region.
Clin Cardiol 1999 Jan
PMID:Efficient in vivo catheter-based pericardial gene transfer mediated by adenoviral vectors. 992 64

Human immunodeficiency virus (HIV, lentivirus) type-1 based vectors have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and long term transgene expression. We used a three-plasmid expression system to generate pseudotyped lentivirus-based vectors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal repeat-directed expression of HIV. Using this system we successfully generated versatile high titer lentivirus at titers of up to 2 x 10(8) transducing units/ml (TU/ml), and improved transduction efficiency in various cell types from seven to over twenty fold. We demonstrate its applicability of these vectors for the efficient transduction of non-dividing cells, including post mitotic beating rat cardiac myocytes and well-differentiated rat L6 myofibers. While both lentivirus-based and murine retrovirus-based vectors effectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myocytes and yielded titers of (6.3 +/- 1.2) x 10(5) TU/ml; however murine retrovirus-based vectors showed low transduction efficiency with titers reaching only (8.9 +/- 2.1) x 10(2) TU/ml. Furthermore, even 12 days after induction of differentiation of L6 myofibers, lentivirus-mediated transduction of beta-galactosidase (beta-Gal) at approximately 30-40% of the maximum expression levels achieved in replicating myoblasts. In contrast, the expression of beta-Gal following transduction of the myofibers by murine retrovirus-based vectors fell to less than 1% of an already reduced level of transduction in undifferentiated confluent myoblasts. These results demonstrate that lentivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an efficient method and provides a new tool for research and therapy for cardiovascular diseases.
J Mol Cell Cardiol 1999 Nov
PMID:A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. 1059 Oct 30

Gene transfer techniques are increasingly being used to study blood vessel biology and develop models for gene therapy. To date, there are no reports of pulmonary vascular gene transfer performed either without adjunctive agents or during angioplasty. We sought to demonstrate the feasibility of recombinant gene transfer to the pulmonary artery of juvenile pigs using naked plasmid DNA delivered via percutaneous angioplasty techniques. Plasmid DNA directing the expression of beta-galactosidase was used to transfect one pulmonary artery while the contralateral vessel served as an untreated control. One delivery technique used a standard angioplasty balloon coated with a DNA-heparin mixture. The second involved infusion of DNA between an angioplasty balloon and a surrounding, microporous balloon. Vessels were harvested 3 or 4 days following gene delivery. Protein expression was demonstrated by immunohistochemical staining in transfected but not control vessels in 9/9 pigs. Vascular wall expression was limited to endothelial cells. Pulmonary artery gene transfer using naked plasmid DNA delivered via percutaneous angioplasty techniques is feasible. Using naked plasmid DNA removes the potential for toxicity associated with adjunctive agents. The described techniques provide novel methods for studying pulmonary vascular biology in vivo and for developing future gene therapies.
Pediatr Cardiol
PMID:Percutaneous pulmonary artery gene transfer in pigs using naked plasmid DNA delivered during angioplasty. 1081 78

We examined the intracellular signaling mechanism for tumor necrosis factor-alpha (TNF-alpha)-induced cardiac hypertrophy in isolated rat neonatal cardiomyocytes. TNF-alpha enhanced the expression of a kappa B-dependent reporter gene construct in a dose-dependent manner, which was transiently transfected in cardiomyocytes. Electrophoretic mobility shift assay demonstrated that TNF-alpha induced nuclear factor- kappa B (NF-kappa B)-specific DNA binding. Cultured cardiomyocytes were infected with a recombinant adenoviral vector expressing a degradation-resistant mutant of I kappa B alpha (AdI kappa B alpha 32/36A). The I kappa B alpha mutant suppressed NF-kappa B activation induced by TNF- alpha. In cardiomyocytes infected with AdI kappa B alpha 32/36A, TNF-alpha-induced hypertrophic responses, including increases in cell size, protein synthesis and atrial natriuretic factor production and enhancement of sarcomeric organization, were remarkably attenuated compared to the cells infected with an adenovirus expressing bacterial beta-galactosidase. Using a reactive oxygen species (ROS)-sensitive fluorescent dye, 2', 7'-dichlorofluorescin, we observed an increase in fluorescent signal in cardiomyocytes over time, upon addition of TNF-alpha. Preincubation of n-acetyl cysteine (NAC), an antioxidant, prior to TNF-alpha treatment, abolished TNF-alpha -induced ROS generation. NAC abolished TNF-alpha-induced NF-kappa B activation and hypertrophic responses. These findings indicated that TNF-alpha-induced cardiomyocyte hypertrophy is mediated through NF-kappa B activation via the generation of ROS.
J Mol Cell Cardiol 2002 Feb
PMID:Involvement of reactive oxygen species-mediated NF-kappa B activation in TNF-alpha-induced cardiomyocyte hypertrophy. 1185 62

Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21(Cip1/Waf1). Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-alpha for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2% vs. 4.3%, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58% of Ras-infected cells stained positive for senescence-associated beta-galactosidase activity as opposed to 2% in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21(Cip1/Waf1) in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-alpha induced-apoptosis. Surprisingly, senescence-associated beta-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21(Cip1/Waf1). Future studies have to investigate a potential role of Ras in human vascular biology.
Basic Res Cardiol 2002 Mar
PMID:Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1). 1200 58

Doxorubicin is a chemotherapeutic agent that can induce cardiotoxicity and congestive heart failure (CHF). In this study we tested whether intracoronary Akt1 gene delivery could inhibit doxorubicin-induced CHF. Saline or a replication defective adenoviral vector expressing constitutively-active Akt1 (myrAkt) or beta-galactosidase (betagal) was delivered to the myocardium of 8 week old rats one day prior to initiating doxorubicin administration. In animals receiving saline or betagal, doxorubicin resulted in significant decreases in cardiac function and retarded post-natal heart growth at the 5 weeks time point. In contrast, transduction of myrAkt protected hearts against doxorubicin-induced decreases in fractional shortening and cardiac index, and improved left ventricular function at 5 weeks time point. Delivery of myrAkt also reversed the doxorubicin-induced reduction in post-natal heart growth and diminished lung edema. These data show that myocardial Akt can inhibit doxorubicin-induced reductions in cardiac function and growth, suggesting that manipulation of this signaling pathway may have utility for the treatment of congestive heart failure.
J Mol Cell Cardiol 2002 Oct
PMID:Elevated myocardial Akt signaling ameliorates doxorubicin-induced congestive heart failure and promotes heart growth. 1239 81


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