Synthesis and preliminary biological evaluation of O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET) as a potential cationic amino acid PET tracer for tumor imaging.

Amino acid transport is an attractive target for oncologic imaging. Despite a high demand of cancer cells for cationic amino acids, their potential as PET probes remains unexplored. Arginine, in particular, is involved in a number of biosynthetic pathways that significantly influence carcinogenesis and tumor biology. Cationic amino acids are transported by several cationic transport systems including, ATB(0,+) (SLC6A14), which is upregulated in certain human cancers including cervical, colorectal and estrogen receptor-positive breast cancer. In this work, we report the synthesis and preliminary biological evaluation of a new cationic analog of the clinically used PET tumor imaging agent O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET), namely O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET). Reference compound and precursor were prepared by multi-step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 16-20% decay-corrected yields with radiochemical purity >99%. The new tracer showed good stability in vitro and in vivo. Cell uptake assays demonstrated that FEMAET and [(18)F]FEMAET accumulate in prostate cancer (PC-3) and small cell lung cancer cells (NCI-H69), with an energy-dependent mechanism. Small animal PET imaging with NCI-H69 xenograft-bearing mice revealed good tumor visualization comparable to [(18)F]FET and low brain uptake, indicating negligible transport across the blood-brain barrier. In conclusion, the non-natural cationic amino acid PET probe [(18)F]FEMAET accumulates in cancer cells in vitro and in vivo with possible involvement of ATB(0,+).

ABC-type amino acid uptake transporters Bgt and N-II of Anabaena sp. strain PCC 7120 share an ATPase subunit and are expressed in vegetative cells and heterocysts.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can fix N(2) in differentiated cells called heterocysts. Anabaena open reading frames alr4167 and alr3187 encode, respectively, an ATPase subunit, BgtA, and a composite protein bearing periplasmic substrate-binding and transmembrane domains, BgtB, of an ABC-type high-affinity basic amino acid uptake transporter (Bgt). Open reading frame alr4167 is clustered with open reading frames alr4164, alr4165 and alr4166 that encode a periplasmic substrate-binding protein, NatF, and transmembrane proteins NatG and NatH respectively. The NatF, NatG, NatH and BgtA proteins constitute an ABC-type uptake transporter for acidic and neutral polar amino acids (N-II). The Bgt and N-II transport systems thus share the ATPase subunit, BgtA. These transporters together with the previously characterized ABC-type uptake transporter for proline and hydrophobic amino acids (N-I) account for more than 98% of the amino acid transport activity exhibited by Anabaena sp. strain PCC 7120. In contrast to N-I that is expressed only in vegetative cells, the Bgt and N-II systems are present in both vegetative cells and heterocysts. Whereas Bgt is dispensable for diazotrophic growth, N-II appears to contribute together with N-I to the diazotrophic physiology of this cyanobacterium.

Comparative gene expression profiles of intestinal transporters in mice, rats and humans.

We have studied gene expression profiles of intestinal transporters in model animals and humans. Total RNA was isolated from duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mice, rats, and humans were about 60% of 22,690 sequences, 40% of 8739, and 47% of 12,559, respectively. A total of 86 genes involving transporters expressed in mice, 50 genes in rats, and 61 genes in humans were detected. Mice exhibited abundant mRNA expressions for peptide transporter HPT1, amino acid transporters CSNU3, CT1 and ASC1, nucleoside transporter CNT2, organic cation transporter SFXN1, organic anion transporter NBC3, glucose transporter SGLT1, and fatty acid transporters FABP1 and FABP2. Rats showed high expression profiles of peptide transporter PEPT1, amino acid transporters CSNU1 and 4F2HC, nucleoside transporter CNT2, organic cation transporter OCT5, organic anion transporter SDCT1, glucose transporter GLUT2 and GLUT5, and folate carrier FOLT. In humans, the highly expressed genes were peptide transporter HPT1, amino acid transporters LAT3, 4F2HC and PROT, nucleoside transporter CNT2, organic cation transporter OCTN2, organic anion transporters NADC1, NBC1 and SBC2, glucose transporters SGLT1 and GLUT5, multidrug resistance-associated protein RHO12, fatty acid transporters FABP1 and FABP2, and phosphate carrier PHC. Overall these data reveal diverse transcriptomic profiles for intestinal transporters among these species. Therefore, this transcriptional data may lead to more effective use of the laboratory animals as a model for oral drug development.

Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes.

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.

Dual-phenotype GABA/glutamate neurons in adult preoptic area: sexual dimorphism and function.

It is generally assumed that the inhibitory neurotransmitter GABA and the stimulatory neurotransmitter glutamate are released from different neurons in adults. However, this tenet has made it difficult to explain how the same afferent signals can cause opposite changes in GABA and glutamate release. Such reciprocal release is a central mechanism in the neural control of many physiological processes including activation of gonadotropin-releasing hormone (GnRH) neurons, the neural signal for ovulation. Activation of GnRH neurons requires simultaneous suppression of GABA and stimulation of glutamate release, each of which occurs in response to a daily photoperiodic signal, but only in the presence of estradiol (E2). In rodents, E2 and photoperiodic signals converge in the anteroventral periventricular nucleus (AVPV), but it is unclear how these signals differentially regulate GABA and glutamate secretion. We now report that nearly all neurons in the AVPV of female rats express both vesicular glutamate transporter 2 (VGLUT2), a marker of hypothalamic glutamatergic neurons, as well as glutamic acid decarboxylase and vesicular GABA transporter (VGAT), markers of GABAergic neurons. These dual-phenotype neurons are the main targets of E2 in the region and are more than twice as numerous in females as in males. Moreover, dual-phenotype synaptic terminals contact GnRH neurons, and at the time of the surge, VGAT-containing vesicles decrease and VGLUT2-containing vesicles increase in these terminals. Thus, we propose a new model for ovulation that includes dual-phenotype GABA/glutamate neurons as central transducers of hormonal and neural signals to GnRH neurons.