Search

Main Conclusion: Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.

Grapevine embryogenic cultures are ideal target tissues for inserting desired traits of interest and improving existing cultivars via precision breeding (PB). PB is a new approach that, like conventional breeding, utilizes only DNA fragments obtained from sexually compatible grapevine plants. Embryogenic culture induction occurs by placing leaves or stamens and pistils on induction medium with a dark/light photoperiod cycle for 12-16 weeks. Resulting cultures produce sectors of embryogenic and non-embryogenic callus, which can be identified on the basis of callus morphology and color. Somatic embryo development occurs following transfer of embryogenic callus to development medium and cultures can be maintained for extended periods of time by transfer of the proliferating proembryonic masses to fresh medium at 4-6-week intervals. To demonstrate plant recovery via PB, somatic embryos at the mid-cotyledonary stage are cocultivated with Agrobacterium containing the desired gene of interest along with a, non-PB, enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Modified cultures are grown on proliferation and development medium to produce uniformly modified somatic embryos via secondary embryogenesis. Modified embryos identified on the basis of green fluorescence and kanamycin resistance are transferred to germination medium for plant development. The resulting plants are considered to prototype examples of the PB approach, since they contain egfp/nptII, a non-grapevine-derived fusion gene. Uniform green fluorescent protein (GFP) fluorescence can be observed in all tissues of regenerated plants.

Facilitated hexose transporters (GLUTs) mediate the transport of hexoses and other substrates across the membranes of numerous cell types, and while some are expressed ubiquitously (e.g., GLUT1), others are more tissue specific (e.g., GLUT5). These properties have been exploited for the imaging of cancer cells by the use of hexose based probes, including fluorinated hexose derivatives for use with positron emission tomography (PET). However, design of new probes has been hampered by a limited understanding of how GLUT transporters interact with their substrates at the molecular level. Two fluorinated fructose surrogates designed for uptake by the GLUT5 transporter are described here: 3-deoxy-3-fluoro-D-fructose (3-FDF) and 1-deoxy-1-fluoro-2,5-anhydromannitol (1-FDAM). Synthesis (both cold and radiolabeled) and in vitro analysis of their transport characteristics in two breast cancer cell lines (EMT-6 and MCF-7) expressing GLUT5 are detailed. Both analogues are readily taken up into both cancer cell lines, with uptake mediated primarily by GLUT5. They also have low IC50 values, indicating a high affinity for the transporter, suggesting that the uptake of these probes would be unaffected by endogenously circulating fructose. Selective uptake by GLUT5 was also demonstrated in Xenopus oocytes. Finally, these results are the first demonstration that a hexose existing predominantly in the pyranose ring structure (3-FDF) is transported by GLUT5, strongly suggesting that this transporter can handle both furanose and pyranose forms of fructose.

Dichlorocyclopropanation of 2-amino-1,3-dienes affords 1-alkenyl-1-amino-2,2-dichlorocyclopropanes which undergo silver-assisted 2-π electrocyclic opening to furnish 3-aminopentadienyl cations. Nazarov-type cyclization of these intermediates leads to cyclopentenone iminium salts, which provide allylic amines upon reduction. This process, the imino version of the traditional Nazarov reaction, can also be combined with an interrupted Nazarov domino process to give polycyclic amines.

Introduction: Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[(18)F]fluoro-D-fructose (6-[(18)F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers., Methods: Uptake of 6-[(18)F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [(18)F]FDG. Biodistribution of 6-[(18)F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [(18)F]FDG. 6-[(18)F]FDF metabolism was investigated in mouse blood and urine., Results: 6-[(18)F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30±4% (n=9) and 12±1% (n=7) ID/mg protein 6-[(18)F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-d-fructose had a 10-fold higher potency than fructose to inhibit 6-[(18)F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65±0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75±0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0.76±0.05; n=3] compared to EMT-6 tumors (SUV=1.23±0.09; n=3). Interestingly, [(18)F]FDG uptake was significantly different in MCF-7 tumors (SUV(15 min) 0.74±0.12 to SUV(120 min) 0.80±0.15; n=3) versus EMT-6 tumors (SUV(15 min) 1.01±0.33 to SUV(120 min) 1.80±0.25; n=3). 6-[(18)F]FDF was shown to be a substrate for recombinant human ketohexokinase, and it was metabolized rapidly in vivo., Conclusion: Based on the GLUT5 specific transport and phosphorylation by ketohexokinase, 6-[(18)F]FDF may represent a novel radiotracer for PET imaging of GLUT5 and ketohexokinase-expressing tumors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The Nazarov reaction entails the electrocyclic closure of a conjugated pentadienyl cation to furnish a cyclopentenyl cation. In its conventional protocol, this intermediate is allowed to undergo elimination through loss of an adjacent proton, providing cyclopentenone products. This feature article describes a relatively new class of domino and cascade processes in which the mechanistic course of the Nazarov reaction is diverted at the point of the cyclopentenyl cation intermediate, collectively referred to as "interrupted Nazarov reactions." A variety of intra- or intermolecular nucleophiles, including silyl hydrides, alkenes, arenes, amines, halides, oxygen nucleophiles and azides, can capture the cation at one terminus, resulting in interesting polycyclic structures. 1,3-Dienes also trap the cyclopentenyl cation, but in this case via concerted [4 + 3]-cycloaddition to give keto-bridged cyclooctenes. An alternative method for generating the pentadienyl cyclization precursor from dichlorocyclopropanes rather than cross-conjugated dienones is also discussed, along with some novel trapping processes.

FDG-based imaging with positron emission tomography (PET) has been widely used in the detection of cancer, but has not reached its full potential. In breast cancer, the glucose/fructose transporter GLUT2 and the fructose transporter GLUT5 are known to be overexpressed in transformed tissues, implicating that a fructose-based analogue would be a useful target for the improved imaging of breast cancer. We have successfully synthesized the fluorinated fructose compound, 6-deoxy-6-fluoro-D-fructose (6FDF) and examined its potential for transport and accumulation in breast cancer cells. Expression analysis of GLUT isoforms was performed on two GLUT5 expressing breast cancer cell lines using western blotting and immunocytochemistry. Uptake and inhibition studies were undertaken using [14C]-labelled hexoses. Transport inhibition studies showed dose dependent inhibition of fructose transport in both cell lines by the newly synthesized 6-deoxy-6-fluoro-D-fructose (6FDF). Also, near linear uptake over time of [14C]-labelled 6FDF was observed in both cell lines. It appears that 6FDF may have great promise for use in in vivo PET imaging of breast cancer. Ongoing work will confirm the efficacy of this compound in imaging in mouse models.

Readily available five- and six-membered lactones and N-sulfonyllactams undergo efficient addition of t-butyl propiolate, and the resulting adducts undergo cycloisomerization to six- and seven-membered cyclic ethers or amines in the presence of pyridinium acetate. The ring expansion process occurs in generally good yields and is proposed to involve a nucleophilic catalysis mechanism.

2-Siloxy-2-alkenyl-1,1-dichlorocyclopropanes with aryl-terminated side chains undergo silver-assisted electrocyclic opening/Nazarov cyclization. The resulting 2-siloxycyclopentenyl cations are intercepted by the pendant arenes to furnish tricyclic adducts in moderate to good yields. In cases where the arene trap was tethered through the cyclopropane unit, a new mode of trapping occurred to generate unique bridged carbon frameworks.

Regioselective dichlorocyclopropanation of 2-silyloxydienes furnishes vinylcyclopropanol silyl ethers in good yield. Treatment with silver(I) at room temperature effects disrotatory electrocyclic opening to a 2-chloro-3-silyloxypentadienyl cation, which then undergoes conrotatory (Nazarov) electrocyclization to provide chlorocyclopentenones. This two-step sequence offers a convenient and mild alternative to the standard Nazarov cyclization protocol via a formal 4+1 construction and furnishes products containing useful halogen functionality. In one case possessing a pendant phenyl group, interrupted Nazarov reaction to give a benzohydrindenone was observed.

[reaction: see text] The reactions of (trans-2-phenylcyclopropyl)ethyne, 1a, (trans,trans-2-methoxy-3-phenylcyclopropyl)ethyne, 1b, and (trans,trans-2-methoxy-1-methyl-3-phenylcyclopropyl)ethyne, 1c, with either aqueous sulfuric acid or tris(trimethylsilyl)silane (or tributyltin hydride) and AIBN have been investigated. Protonation and addition of the silyl (or stannyl) radical occurred at the terminal position of the alkyne giving an alpha-cyclopropyl-substituted vinyl cation or radical, respectively. Under both reaction conditions, 1a yielded products derived from ring opening toward the phenyl substituent. Alkynes 1b and 1c, however, gave different products depending on whether radical or cationic conditions were used. When radical conditions were employed, products derived from regioselective ring opening toward the phenyl substituent were obtained. In contrast, when cationic conditions were employed, products derived from selective ring opening toward the methoxy substituent were isolated. The corresponding alpha-cyclopropyl-substituted vinyllithium derivatives were also synthesized and were found to be stable toward rearrangement. An estimate of the rate constants for ring opening of the alpha-cyclopropylvinyl cations was also made: values of 10(10)-10(12) s(-1) were found for the vinyl cations derived from protonation of the terminal carbon of alkynes 1a-c. Based on these results, cyclopropyl alkynes 1a-c can be classified as hypersensitive mechanistic probes for the detection of vinyl radical or cationic intermediates generated adjacent to the cyclopropyl ring and, in the case of 1b and 1c, the distinction between a radical or cationic intermediate is possible.

This study investigated the relationship of paternal masculinity-femininity to filial creativity in a sample of high school students and their fathers. Thirty-one male high school students were identified as creative on the basis of teacher nominations and scores on the Remote Associates Test. An equal size control group of students matched on sex, educational level, grade point average, and geographical residence was formed. Fathers of both experimental and control students were administered a battery of tests which included a variety of masculinity-femininity measures. The results seem to support the general hypothesis that creativity is related to parental identification as a function of a less conventional sex-role stereotype, and the more specific hypothesis that there is a relation between paternal masculinity-femininity and filial creativity.