A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates

Diviacco, Silvia; Norio, Paolo; Zentilin, Lorena; Menzo, Stefano; Clementi, Massimo; Biamonti, Giuseppe; Riva, Silvano; Falaschi, Arturo; Giacca, Mauro

doi:10.1016/0378-1119(92)90220-j

Cited by 247 publications

(182 citation statements)

References 23 publications

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“…rAAV titres were determined by measuring the copy number of viral genomes in pooled, dialyzed gradient fractions. This was achieved by a competitive PCR procedure [32] using primers and competitors mapping in the CMV promoter region common to all vectors. The purified viral preparations used for this work had particle titres of about 1×10 12 viral genomes per ml.…”

Section: Methodsmentioning

confidence: 99%

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse

et al. 2003

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Aims/hypothesis. We studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates (db+/+m). Methods. Animals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds. Results. We found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strenght of the wound and enhanced the wound content of VEGF. Conclusion/interpretation. Our study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes. [Diabetologia (2003) 46:546-555]

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Section: Methodsmentioning

confidence: 99%

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse

et al. 2003

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“…48 rAAV titers were determined by measuring the copy number of viral genomes in pooled, dialyzed gradient fractions. This was achieved by a competitive PCR procedure, 49 using primers and competitors mapping in the CMV promoter region common to all vectors. 50 The purified viral preparations used for this work had particles titers of approximately 1 × 10 12 viral genomes per ml.…”

Section: Materials and Methods Raav Vector Preparation And Characterimentioning

confidence: 99%

Recombinant AAV vector encoding human VEGF165 enhances wound healing

et al. 2002

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“…For data analysis, the logarithm of the ratio of the density (A 1 /construct) was plotted as a function of the log of the concentration of the construct and fitted by linear regression. The 0 value of the log of the ratio (A 1 /construct) represents the point at which the A 1 PCR product and the construct product are present in equal amounts and therefore are also comparable before the PCR reaction (Diviacco et al 1992;Galea and Feinstein 1992). …”

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Interleukin-6 Enhances Expression of Adenosine A1 Receptor mRNA and Signaling in Cultured Rat Cortical Astrocytes and Brain Slices

Biber

2001

Neuropsychopharmacology

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The inhibitory neuromodulator adenosine is released in the brain in high concentrations under conditions of exaggerated neuronal activity such as ischemia and seizures, or electroconvulsive treatment. By inhibiting neural overactivity, adenosine counteracts seizure activity and promotes neuronal survival. Since stimulation of adenosineAdenosine, as a metabolite of ATP, nature's general energy source, has acquired early in evolution the general function of signaling and counteracting a dysbalance of energy supply and demand (Newby 1984). In the brain, adenosine acts as an inhibitory neuromodulator, which is released under conditions of neuronal activity and reduces this activity by inhibition of transmitter release and postsynaptic hyperpolarisation. Brain damage, e.g., by stroke, ischemia, and seizures leads to a pronounced increase in extracellular adenosine (for review see Rudolphi and Schubert 1996) which counteracts seizure activity (Dragunow 1988) and promotes neuronal survival (Deckert and Gleiter 1994;Picano and Abbracchio 1998;Dux et al. 1990).In addition to its role as an endogenous anticonvulsant and neuroprotective agent, adenosine is now recognized as an important regulator of sleep and wakefulness (for review see Porkka-Heiskanen 1999 (van Calker et al. 1978(van Calker et al. , 1979, but more recent findings have revealed coupling of adenosine receptors to other signaling systems including the phosphoinositol system, potassium, and calcium channels (for review see Fredholm et al. 1994;Ralevic and Burnstock 1998). Adenosine mediates neuroprotection by inhibiting presynaptically the release of several neurotransmitters including the excitatory neurotransmitter glutamate (Ribeiro 1995). Postsynaptically, adenosine increases conductances of various K ϩ -and Cl Ϫ -channels, which hyperpolarize the membrane potential and counteract a transmitter induced depolarisation with a subsequent reduction of Ca 2 ϩ -influx and stabilization of the Mg 2 ϩ blockage of NMDA receptors (Rudolphi et al. 1992a,b;Rudolphi and Schubert 1996). Most, if not all, of these actions of adenosine are mediated via the adenosine A 1 receptor, whereas the role of the other adenosine receptor subtype in neuroprotection is less clear (Schubert et al. 1997;Abbracchio and Cattabeni 1999;Heurteaux et al. 1995). Accordingly, upregulation of adenosine A 1 receptors by chronic treatment with A 1 -antagonists increases the neuroprotective effect of adenosine (Sutherland et al. 1991; Rudolphi et al. 1992a,b).Upregulation of adenosine A1-receptors is also observed after treatments that exert antidepressive effects in humans such as seizures and electroconvulsive treatment (ECT) (Newman et al. 1984;Gleiter et al. 1989;Angelatou et al..1993;Pagonopoulou et al. 1993), REM sleep deprivation (Yanik and Radulovacki 1987), and chronic treatment with carbamazepine (Daval et al. 1989;Biber et al. 1999;van Calker et al. 2000). While the upregulation by carbamazepine of adenosine A 1 receptors is readily comprehensible from carbamazepine's selective antag...

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A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates

Cited by 247 publications

References 23 publications

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse

Recombinant AAV vector encoding human VEGF165 enhances wound healing

Interleukin-6 Enhances Expression of Adenosine A1 Receptor mRNA and Signaling in Cultured Rat Cortical Astrocytes and Brain Slices

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