Mediated internalization of folates is required for cellular macromolecular biosynthesis. Multiple carrier-mediated mechanisms have been identified that can fulfill this role in a variety of mammalian cell types, including neoplastic cells, with and without proliferative potential. The absorption of dietary folates also relies on the function of a carrier-mediated system in mature luminal epithelium of small intestine. The various carrier-mediated systems can be distinguished by their preferences for various folate compounds as permeants as well as by differences in temperature and pH dependence. The widely studied one-carbon, reduced-folate transport system is mediated by a transporter encoded by the newly discovered RFC-1 (reduced-folate carrier) gene. The characteristics of this gene in rodent and human cells are similar, consistent with the close similarity between these species of folate transport mediated by this transporter. However, differences occur in the form of tissue-specific expression, alternate splicing, and 5' end mRNA heterogeneity, as well as in promoter utilization regulating transcription. RFC-1 gene expression also appears to regulate luminal epithelial cell folate absorption in small intestine. However, the properties of RFC-1-mediated folate transport in these cells is anomalous when compared with that seen in nonabsorptive cell types. Detailed mechanisms as to the regulation of RFC-1 transcription are now emerging along with other information on structure and function of the transporter and its alteration following mutation.

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PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with 125I, or with 111In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from 111In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from 125I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, 111In-labelled and 125I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with 111In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-014-2940-2) contains supplementary material, which is available to authorized users.

Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidation of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by ␤-hydroxyaspartate and cysteic acid but not by ␣-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltP Bsu ) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was determined. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon. gltP Bsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compounds such as aspartate, fumarate, malate, and succinate. Both sodium-and proton-coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the family of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.The amino acid transporters in the thermophile Bacillus stearothermophilus studied to date facilitate an electrogenic symport reaction in which Na ϩ is used as the coupling ion. The apparent affinity constants for Na ϩ are in the range of 0.5 to 1 mM (14). The transport of glutamate and aspartate is driven by the proton motive force (⌬p) but also by an inwardly directed Na ϩ gradient (⌬pNa). The transport of glutamate occurs most likely in symport with one H ϩ and one Na ϩ (7); the apparent affinity constant for Na ϩ is Ͻ10 M. So far, sodium/proton/ glutamate transporters have been found in the thermophiles Bacillus sp. strain IS1 (gltT Bi ) (42), B. stearothermophilus (gltT Bs ), and Bacillus caldotenax (gltT Bc ). The genes encoding GltT Bs and GltT Bc have been cloned and functionally expressed in Escherichia coli (43).Studies on the transport of L-glutamate and L-aspartate in whole cells of B. subtilis W23, 60015, 6GM, and 8G5 suggested that ⌬pNa is not involved as a driving force in this mesophilic Bacillus species (41). The glutamate transporter of B. subtilis is likely to differ from those of thermophilic bacilli with respect to not only cation selectivity but also thermostability. In order to compare the sodium/proton/glutamate symport protein of the thermophile B. stearothermophilus with the glutamate transport protein of the closely related mesophile B. subtilis, the latter system was studied at the molecular level. This study confirms that glutamate uptake in B. subtilis is indeed coupled to th...

Mediated folate compound transport inward in isolated luminal epithelial cells from mouse small intestine was delineated as pH-dependent and non-pH-dependent components on the basis of their differential sensitivity to the stilbene inhibitor, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid. pH dependence was manifested as higher maximum capacity (Vmax) for influx of l, L-5-CH3-H4folate at acidic pH compared with neutral or alkaline pH with no effect on saturability (Km). The pH-dependent component was relatively insensitive to inhibition by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and highly saturable (Km or Ki = 2 to 4 microM) in the case of folic acid, folate coenzymes, and 4-aminofolate analogues as permeants or inhibitors. The non-pH-dependent component was highly sensitive to 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid and poorly and variably saturable (Km or Ki = 20 to >2000 microM) with respect to these folate compounds. Only the pH-dependent transport component was developmentally regulated, showing much higher maximum capacity for l,L-5-CH3-H4folate influx in mature absorptive rather than proliferative crypt cells. The increase in pH-dependent influx during maturation was associated with an increase in RFC-1 gene expression in the form of a 2.5-kilobase RNA transcript and 58-kDa brush-border membrane protein detected by folate-based affinity labeling and with anti-mouse RFC-1 peptide antibodies. The size of this protein was the same as that encoded by RFC-1 mRNA. The treatment of mature absorptive cells with either the affinity label or the anti-RFC-1 peptide antibodies inhibited influx of l, L-[3H]-5-CH3-H4folate in a concentration-dependent manner. These results strongly suggest that pH-dependent folate absorption in this tissue is regulated by RFC-1 gene expression.

Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in non-radical resections, increased cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastro-intestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA-targeted near-infrared fluorescent (NIRF) tracer, based on a disulphide stabilized single-chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell-based plate assays and orthotopic colorectal (HT-29, well differentiated) and pancreatic (BXPC-3, poorly differentiated) xenogeneic human-mouse models. NIRF signals were visualized using the clinically compatible FLARE™ imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor-to-background ratios of 5.1±0.6 at 72 h post-injection, which proved suitable for intra-operative detection and delineation of tumor boarders and small (residual) tumor-nodules in mice, between 8 h and 96 h post-injection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor-specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent-guided surgery applications. If successful translated clinically, this tracer could help improve the completeness of surgery and thus survival.

This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.

The magnetic properties and safety of dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) have facilitated their clinical use as MRI contrast agents and stimulated research on applications for SPIONs in particle imaging and magnetic hyperthermia. The wider clinical potential of SPIONs, however, has been limited by their rapid removal from circulation via the reticuloendothelial system (RES). We explored the possibility of extending SPION circulatory time using fucoidan, a seaweed-derived food supplement, to inhibit RES uptake. The effects of fucoidan on SPION biodistribution were evaluated using ferucarbotran, which in its pharmaceutical formulation (Resovist) targets the RES. Ferucarbotran was radiolabeled at the iron oxide core with technetium-99m (Tc; t = 6 h) or zirconium-89 (Zr; t = 3.3 days). Results obtained with Tc-ferucarbotran demonstrated that administration of fucoidan led to a 4-fold increase in the circulatory half-life (t slow) from 37.4 to 150 min (n = 4; P < 0.0001). To investigate whether a longer circulatory half-life could lead to concomitant increased tumor uptake, the effects of fucoidan were tested with Zr-ferucarbotran in mice bearing syngeneic subcutaneous (GL261) tumors. In this model, the longer circulatory half-life achieved with fucoidan was associated with a doubling in tumor SPION uptake (n = 5; P< 0.001). Fucoidan was also effective in significantly increasing the circulatory half-life of perimag-COOH, a commercially available SPION with a larger hydrodynamic size (130 nm) than ferucarbotran (65 nm). These findings indicate successful diversion of SPIONs away from the hepatic RES and show realistic potential for future clinical applications.