Functional mutants of Azospirillum brasilense elicit beneficial physiological and metabolic responses in Zea mays contributing to increased host iron assimilation.

Iron (Fe), an essential element for plant growth, is abundant in soil but with low bioavailability. Thus, plants developed specialized mechanisms to sequester the element. Beneficial microbes have recently become a favored method to promote plant growth through increased uptake of essential micronutrients, like Fe, yet little is known of their mechanisms of action. Functional mutants of the epiphytic bacterium Azospirillum brasilense, a prolific grass-root colonizer, were used to examine mechanisms for promoting iron uptake in Zea mays. Mutants included HM053, FP10, and ipdC, which have varying capacities for biological nitrogen fixation and production of the plant hormone auxin. Using radioactive iron-59 tracing and inductively coupled plasma mass spectrometry, we documented significant differences in host uptake of Fe2+/3+ correlating with mutant biological function. Radioactive carbon-11, administered to plants as 11CO2, provided insights into shifts in host usage of 'new' carbon resources in the presence of these beneficial microbes. Of the mutants examined, HM053 exhibited the greatest influence on host Fe uptake with increased plant allocation of 11C-resources to roots where they were transformed and exuded as 11C-acidic substrates to aid in Fe-chelation, and increased C-11 partitioning into citric acid, nicotianamine and histidine to aid in the in situ translocation of Fe once assimilated.

Investigations of acetonitrile solvent cluster formation in supercritical carbon dioxide, and its impact on microscale syntheses of carbon-11-labeled radiotracers for PET.

A new strategy has been developed for synthesizing positron emission tomography (PET) radiotracers using [11C]methyl iodide. This strategy relies on the ability of organic co-solvents to cluster within mixtures of supercritical fluids resulting in localized regions of high density which can serve as microscopic pockets for reaction. We've shown that acetonitrile will cluster about dilute solutes when mixtures of this co-solvent with carbon dioxide are forced to behave as a homogeneous fluid at the critical point. We applied this strategy in a systematic investigation of the conditions for optimized reaction between methyl iodide and L-alpha-methyl-N-2-propynyl phenethylamine (nordeprenyl) to yield L-deprenyl. Variables such as temperature, ultraviolet light exposure, co-solvent concentration, system pressure, and methyl iodide concentration were explored. The synthesis of radioactive [11C]-L-deprenyl using no-carrier-added concentrations of [11C]methyl iodide was also tested. Results showed that greater than 90% radiochemical yield of the desired product could be attained using 40 times less labeling substrate than in conventional PET tracer syntheses.

Opioid receptor imaging and displacement studies with [6-O-[11C] methyl]buprenorphine in baboon brain.

Buprenorphine (BPN) is a mixed opiate agonist-antagonist used as an analgesic and in the treatment of opiate addiction. We have used [6-O-[11C]methyl]buprenorphine ([11C]BPN) to measure the regional distribution in baboon brain, the test-retest stability of repeated studies in the same animal, the displacement of the labeled drug by naloxone in vivo, and the tissue distribution in mice. The regional distribution of radioactivity in baboon brain determined with PET was striatum > thalamus > cingulate gyrus > frontal cortex > parietal cortex > occipital cortex > cerebellum. This distribution corresponded to opiate receptor density and to previously published data (37). The tracer uptake in adult female baboons showed no significant variation in serial scans in the same baboon with no intervention in the same scanning session. HPLC analysis of baboon plasma showed the presence of labeled metabolites with 92% +/- 2.2% and 43% +/- 14.4% of the intact tracer remaining at 5 and 30 min, respectively. Naloxone, an opiate receptor antagonist, administered 30-40 min after tracer injection at a dose of 1.0 mg/kg i.v., reduced [11C]BPN binding in thalamus, striatum, cingulate gyrus, and frontal cortex to values 0.25 to 0.60 of that with no intervention. There were minimal (< 15%) effects on cerebellum. Naloxone treatment significantly reduced the slope of the Patlak plot in receptor-containing regions. These results demonstrate that [11C]BPN can be displaced by naloxone in vivo, and they affirm the feasibility of using this tracer and displacement methodology for short-term kinetics studies with PET. Mouse tissue distribution data were used to estimate the radiation dosimetry to humans. The critical organ was the small intestine, with a radiation dose estimate to humans of 117 nrad/mCi.

Species differences in [11C]clorgyline binding in brain.

[11C]Clorgyline selectively binds to MAO A in the human brain. This contrasts with a recent report that [11C]clorgyline (in contrast to other labeled MAO A inhibitors) is not retained in the rhesus monkey brain [4]. To explore this difference, we compared [11C]clorgyline in the baboon brain before and after clorgyline pretreatment and we also synthesized deuterium substituted [11C]clorgyline (and its nor-precursor) for comparison. [11C]Clorgyline was not retained in the baboon brain nor was it influenced by clorgyline pretreatment or by deuterium substitution, contrasting to results in humans. This suggests a species difference in the susceptibility of MAO A to inhibition by clorgyline and represents an unusual example of where the behavior of a radiotracer in the baboon brain does not predict its behavior in the human brain.

Methylphenidate decreases regional cerebral blood flow in normal human subjects.

To assess the effects of methylphenidate (MP) on cerebral blood flow (CBF), 5 healthy males were studied using 15O-water and positron emission tomography before and after MP (0.5 mg/kg iv). MP significantly decreased whole brain CBF at 5-10 minutes (25 +/- 11%) and at 30 minutes (20 +/- 10%) after its administration. Decrements in CBF were homogeneous throughout the brain (regional decrements 23-30%) and probably reflect the vasoactive properties of MP. The vasoactive properties of MP should be considered when prescribing this drug chronically and/or when giving it to subjects with cerebrovascular compromise.

Study of brain uptake and biodistribution of [11C]toluene in non-human primates and mice.

Inhalant abuse is a rapidly growing health problem particularly among adolescents. Yet we know little about the neural mechanisms underlying the abuse liability of inhalants, particularly when compared to other addictive drugs. Specifically, our understanding of the relationship between the regional brain phamacokinetics and features classically associated with drug reinforcement is lacking. Under the hypothesis that the abuse liability of toluene can be related to its pharmacokinetic properties and the pattern of regional brain uptake, we developed the methodology for radiolabeling and purifying [11C]toluene for use in PET studies. Here we report the regional brain distribution and kinetics of the widely abused solvent toluene in non-human primates and the whole body biodistribution in mice. To our knowledge, this is the first reported study of the in vivo brain pharmacokinetics of labeled toluene in non-human primates. Rapid uptake of radioactivity into striatal and frontal regions was followed by rapid clearance from the brain. Concurrent findings in rodents indicate similar radio-tracer kinetics, with excretion through kidneys and liver. Taken together, our data provides insight into pharmacokinetic features possibly associated with the abuse liability of toluene.

Remote processing, delivery and injection of H2[15O] produced from a N2/H2 gas target using a simple and compact apparatus.

We report here a simple apparatus for remote trapping and processing of H2[15O] produced from the N2/H2 target. The system performs a three step operation for H2[15O] delivery at the PET imaging facility which includes the following: (i) collecting the radiotracer in sterile water; (ii) adjusting preparation pH through removal of radiolytically produced ammonia, while at the same time adjusting solution isotonicity; and (iii) delivery of the radiotracer preparation to the injection syringe in a sterile and pyrogen-free form suitable for human studies. The processing apparatus is simple, can be remotely operated and fits inside a Capintec Dose Monitoring Chamber for direct measurement of accumulated radioactivity. Using this system, 300 mCi of H2[15O] (15 microA of 8 MeV D+ on target) is transferred from target through 120 m x 3.18 mm o.d. Impolene tubing to yield 100 mCi of H2[15O] which is isotonic, neutral and suitable for human studies. A remote hydraulically driven system for i.v. injection of the H2[15O] is also described. The device allows for direct measurement of syringe dose while filling, and for easy, as well as safe transport of the injection syringe assembly to the patient's bedside via a shielded delivery cart. This cart houses a hydraulic piston that allows the physician to "manually" inject the radiotracer without directly handling the syringe.

Rapid radiochemical and chemical quality control of [11C]putrescine.

A short (4.6 x 50 mm) cation exchange column was used in conjunction with conductivity and radioactivity detectors to determine the radiochemical purity (> 99%) and specific radioactivity (0.5-1.0 Ci/mu mol) of [1-11C]putrescine prepared via Michael addition of [11C]cyanide to acrylonitrile. The absence of acrylonitrile, a rodent carcinogen, from the final preparation was verified at the 50 ng level capillary vapor-phase chromatography (VPC) using a nitrogen-phosphorus detector. Routine VPC analysis using a Poropak Q column and flame ionization detection showed that preparations contained no more than 1 microgram of acrylonitrile.

A universal water target loading system with direct in-target liquid level sensing.

A reliable remote water target loading system has been designed and fabricated around the operation of a single pneumatic syringe dispenser that accesses one of two water reservoirs, filling one or more targets depending on the system configuration. For multiple radionuclide configuration, reservoirs are filled with 18O-enriched water for [18F]fluoride production, and natural abundance water for [13N]nitrate and [13N]nitrite production. When actuated, the system withdraws a calibrated volume of water from a selected reservoir, and automatically dispenses that charge through a short transfer line to the appropriate target. A second actuation then forces the dispenser to purge itself of residual water by dispensing a charge of helium gas through the same line thus completing the transfer of water to the target volume. Direct liquid level sensing inside the target, utilizing the specific resistance response of water, signals the operator that the target is loaded. For single radionuclide configuration, one or both reservoirs can be used with the same type of water, while dispensing is directed to a single target.

An improved synthesis of the inert, diffusible blood-flow tracer, [18F]fluoromethane.

An improved procedure for producing [18F]fluoromethane ([18F]FM) in batches of several hundred mCi is reported. 18F prepared by the 18O (p, n) 18F reaction in a H2(18)O target is trapped on a small column of Dowex 1 (x 10) resin to allow recovery of H2(18)O. One new feature is elution of 18F- from the column with 3 mL of CH3CN containing 67 microL 1.5 M aqueous (Bu)4N+ OH-, after residual H2(18)O has been removed with dry CH3CN. This 18F- solution reacts with CH3I in the presence of Ag2O directly to give [18F]FM, which is swept out of the reaction vessel with a stream of air, from which CH3I and other vapors are removed with a C18 SEP-PAK at room temperature. Another new technique is trapping of [18F]FM on a second SEP-PAK cooled in ethanol/dry ice. After warming the SEP-PAK to room temperature, the trapped [18F]FM can be recovered with either H2O or air. The improvements speed the preparation and minimize hands-on operations. The product has no detectable radiochemical impurities, and a specific activity of greater than 1 Ci/mumol. Non-radioactive CH3CN, CH3I and CH3OH are present at less than 0.2 mumol per batch.

GABAergic inhibition of endogenous dopamine release measured in vivo with 11C-raclopride and positron emission tomography.

Extensive neuroanatomical, neurophysiological, and behavioral evidence demonstrates that GABAergic neurons inhibit endogenous dopamine release in the mammalian corpus striatum. Positron emission tomography (PET) studies in adult female baboons, using the dopamine D2-specific radiotracer 11C-raclopride, were undertaken to assess the utility of this imaging technique for measuring these dynamic interactions in vivo. 11C-raclopride binding was imaged prior to and following the administration of either gamma-vinyl-GABA (GVG), a specific suicide inhibitor of the GABA-catabolizing enzyme GABA transaminase, or lorazepam, a clinically prescribed benzodiazepine agonist. Striatal 11C-raclopride binding increased following both GVG and lorazepam administration. This increase exceeded the test/retest variability of 11C-raclopride binding observed in the same animals. These findings confirm that changes in endogenous dopamine concentrations resulting from drug-induced potentiation of GABAergic transmission can be measured with PET and 11C-raclopride. Finally, this new strategy for noninvasively evaluating the functional integrity of neurophysiologically linked transmitter systems with PET supports its use as an approach for assessing the multiple mechanisms of drug action and their consequences in the human brain.