Nagichettiar Satyamurthy

Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction

The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2′-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind [3H]spiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent [3H]spiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene. Gene Therapy (2001) 8, 1490–1498.

Quantitative imaging of gene induction in living animals

Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D2R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r2 = 0.98) between ‘target’ and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.

Monitoring adenoviral DNA delivery, using a mutant herpes simplex virus type 1 thymidine kinase gene as a PET reporter gene.

Current gene therapy protocols often suffer from an inability to monitor the site, level and persistence of gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a reporter gene for in vivo imaging of reporter gene expression. The presence of the HSV1-tk reporter gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET reporter gene system, both alternative substrates and mutations in the HSV1-tk gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[18F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET reporter gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.

Halogen exchange reactions between alkyl halides and aqueous hydrogen halides. A new method for preparation of alkyl halides.

Abstract Alkyl fluorides and chlorides are efficiently converted to their corresponding alkyl bromides and iodides by using readily available aqueous HBr and HI. The application of the same method for the conversion of some alkyl fluorides to the corresponding alkyl chlorides with conc. HCl is also described.

Efficient conversion of 1-aryl-3, 3-dialkyltriazenes to phenols and oxygen-18 labeled phenols

Abstract Phenols have been synthesized in excellent yields using a cation exchange resin (H + form) assisted decomposition of 1-aryl-3,3-dialkyltriazanes in the presence of water. This method is also amenable to a high yield and high enrichment synthesis of oxygen-17 and 18 labeled phenols.

Memory and Brain Amyloid and Tau Effects of a Bioavailable Form of Curcumin in Non-Demented Adults: A Double-Blind, Placebo-Controlled 18-Month Trial

OBJECTIVEnBecause curcumins anti-inflammatory properties may protect the brain from neurodegeneration, we studied its effect on memory in non-demented adults and explored its impact on brain amyloid and tau accumulation using 2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile positron emission tomography (FDDNP-PET).nnnMETHODSnForty subjects (age 51-84 years) were randomized to a bioavailable form of curcumin (Theracurmin® containing 90u2009mg of curcumin twice daily [Nu2009=u200921]) or placebo (Nu2009=u200919) for 18 months. Primary outcomes were verbal (Buschke Selective Reminding Test [SRT]) and visual (Brief Visual Memory Test-Revised [BVMT-R]) memory, and attention (Trail Making A) was a secondary outcome. FDDNP-PET signals (15 curcumin, 15 placebo) were determined in amygdala, hypothalamus, medial and lateral temporal, posterior cingulate, parietal, frontal, and motor (reference) regions. Mixed effects general linear models controlling for age and education, and effect sizes (ES; Cohens d) were estimated.nnnRESULTSnSRT Consistent Long-Term Retrieval improved with curcumin (ESu2009=u20090.63, pu2009=u20090.002) but not with placebo (ESu2009=u20090.06, pu2009=u20090.8; between-group: ESu2009=u20090.68, pu2009=u20090.05). Curcumin also improved SRT Total (ESu2009=u20090.53, pu2009=u20090.002), visual memory (BVMT-R Recall: ESu2009=u20090.50, pu2009=u20090.01; BVMT-R Delay: ESu2009=u20090.51, pu2009=u20090.006), and attention (ESu2009=u20090.96, pu2009<u20090.0001) compared with placebo (ESu2009=u20090.28, pu2009=u20090.1; between-group: ESu2009=u20090.67, pu2009=u20090.04). FDDNP binding decreased significantly in the amygdala with curcumin (ESu2009=u2009-0.41, pu2009=u20090.04) compared with placebo (ESu2009=u20090.08, pu2009=u20090.6; between-group: ESu2009=u20090.48, pu2009=u20090.07). In the hypothalamus, FDDNP binding did not change with curcumin (ESu2009=u2009-0.30, pu2009=u20090.2), but increased with placebo (ESu2009=u20090.26, pu2009=u20090.05; between-group: ESu2009=u20090.55, pu2009=u20090.02).nnnCONCLUSIONSnDaily oral Theracurmin may lead to improved memory and attention in non-demented adults. The FDDNP-PET findings suggest that symptom benefits are associated with decreases in amyloid and tau accumulation in brain regions modulating mood and memory.

Concepts and Techniques Used in Metabolic Tracer Studies

Studies of ammonia, amino acid, fatty acid, and glucose metabolism have relied primarily on the use of 15N, 14C, 13C, and 3H-labeled compounds. However, tissue sampling and analysis of enriched metabolites, i.e., 15N and 13C by mass spectrometry [1] and NMR [2–4], and 14C and 3H by liquid scintillation counting, limit the application of these techniques for the noninvasive determination of in vivo tissue tracer kinetics.