NOVEL AMINO ACID TRANSPORTER-TARGETED RADIOTRACERS FOR BREAST CANCER IMAGING

Breast cancer is the most common malignancy among women in the world. Its 5-year survival rate ranges from 23.4% in patients with stage IV to 98% in stage I disease, highlighting the importance of early detection and diagnosis. 18F-2-Fluoro-2-deoxy-glucose (18F-FDG), using positron emission tomography (PET), is the most common functional imaging tool for breast cancer diagnosis currently. Unfortunately, 18F-FDG-PET has several limitations such as poorly differentiating tumor tissues from inflammatory and normal brain tissues. Therefore, 18F-labeled amino acid-based radiotracers have been reported as an alternative, which is based on the fact that tumor cells uptake and consume more amino acids to sustain their uncontrolled growth. Among those radiotracers, 18F-labeled tyrosine and its derivatives have shown high tumor uptake and great ability to differentiate tumor tissue from inflammatory sites in brain tumors and squamous cell carcinoma. They enter the tumor cells via L-type amino acid transporters (LAT), which were reported to be highly expressed in many cancer cell lines and correlate positively with tumor growth. Nevertheless, the low radiosynthesis yield and demand of an on-site cyclotron limit the use of 18F-labeled tyrosine analogues. In this study, four Technetium-99m (99mTc) labeled tyrosine/ AMT (α-methyl tyrosine)-based radiotracers were successfully synthesized and evaluated for their potentials in breast cancer imaging. In order to radiolabel tyrosine and AMT, the chelators N,N’-ethylene-di-L-cysteine (EC) and 1,4,8,11-tetra-azacyclotetradecane (N4 cyclam) were selected to coordinate 99mTc. These chelators have been reported to provide stable chelation ability with 99mTc. By using the chelator technology, the same target ligand could be labeled with different radioisotopes for various imaging modalities for tumor diagnosis, or for internal radionuclide therapy in future. Based on the in vitro and in vivo evaluation using the rat mammary tumor models, 99mTc-EC-AMT is considered as the most suitable radiotracer for breast cancer imaging overall, however, 99mTc-EC-Tyrosine will be more preferred for differential diagnosis of tumor from inflammation.

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Synthesis and properties of heterobifunctional photoaffinity reagents for the identification and isolation of receptors

A method employing isotopically- and photoaffinity-labeled probes and polyclonal and monoclonal antibody to the probes for the identification, isolation and recovery of protein receptors is described. Antibody was raised against N-(3-(p-azido-m-($\sp{125}$I) -iodophenyl)) propionate (AIPP) coupled to and photolyzed to BSA. The antibodies specifically bound AIPP-derivatized proteins. An isolation system was developed utilizing this probe and two antigenically identical reversible analogues. N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl)propionyl)amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) reacts with primary amines and N-(((3-p-azido-m-($\sp{125}$I) -iodophenyl)propionyl)amidoethyl)dithiopyridine ($\sp{125}$I-AIPP-PDA) reacts with reduced thiols. The applicability of the system was established by derivatizing known ligands (Transferrin and Interferon-alpha) with one of the probes. The ligand-probe was then allowed to interact with its receptor by incubation with SS5 lymphoma cells and cross-linked by photolysis at 300 nm. The photolyzed ligand/probe/receptor preparation was then recovered with AIPP antibody. Utilization of N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl-propionyl)-amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) allowed the components of the photolyzed complex to be separated by treatment with 2-mercaptoethanol in the SDS-PAGE solubilization buffer. Ligand and receptor labeling were then assessed by Coomassie staining and autoradiography. Results of receptor assays suggest that $\sp{125}$I-AIPP was, indeed, transferred to moieties that represent the receptors for both Transferrin and Interferon-alpha. ^

Long-term synaptic facilitation of tail sensorimotor connections in {\it Aplysia\/}

Long-term sensitization in Aplysia is a well studied model for the examination of the cellular and molecules mechanisms of long-term memory. Several lines of evidence suggest long-term sensitization is mediated at least partially by long-term synaptic facilitation between the sensory and motor neurons. The sensitization training and one of its analogues, serotonin (5-HT), can induce long-term facilitation. In this study, another analogue to long-term sensitization training has been developed. Stimulation of peripheral nerves of pleural-pedal ganglia preparation induced long-term facilitation at both 24 hr and 48 hr. This is the first report that long-term facilitation in Aplysia persists for more than 24 hr, which is consistent with the observation that long-term sensitization lasts for more than one day. Thus, the data support the hypothesis that long-term facilitation is an important mechanism for long-term sensitization.^ One of the major differences between short-term and long-term facilitation is that long-term facilitation requires protein synthesis. Therefore, the effects of anisomycin, a protein synthesis inhibitor, on long-term facilitation was examined. Long-term facilitation induced by nerve stimulation was inhibited by 2 $\mu$M anisomycin, which inhibits $\sim$90% of protein synthesis. Nevertheless, at higher concentration (20 $\mu$M), anisomycin induced long-term facilitation by itself, which raises an interesting question about the function of anisomycin other than protein synthesis inhibition.^ Since protein synthesis is critical for long-term facilitation, a major goal is to identify and functionally characterize the molecules whose mRNA levels are altered during the formation of long-term facilitation. Behavioral training or its analogues (nerve stimulation and 5-HT) increases the level of mRNA of calmodulin (CaM). Thus, the role of Ca$\sp{2+}$-CaM-dependent protein kinase II (CaMKII), a major substrate of CaM, in long-term facilitation induced by nerve stimulation was examined. KN-62, a specific CaMKII inhibitor, did not block either the induction or the maintenance of long-term facilitation induced by nerve stimulation. These data indicate that CaMKII may not be involved in long-term facilitation. Another protein whose mRNA level of a molecule was increased by the behavioral training and the treatment of 5-HT is Aplysia tolloid/BMP-1-like protein 1 (apTBL-1). Tolloid in Drosophila and BMP-1 in human tissues are believed to be secreted as a metalloprotease to activate TGF-$\beta.$ Thus, the long-term effects of recombinant human TGF-$\beta1$ on synaptic strength were examined. Treatment of ganglia with TGF-$\beta1$ produced long-term facilitation, but not short-term or intermediate-term facilitation ($\le$4 hr). In addition, TGF-$\beta1$ and 5-HT were not additive in producing long-term facilitation, which indicates an interaction between two cascades. Moreover, 5-HT-induced facilitation (at both 24 hr and 48 hr) and nerve stimulation-induced facilitation (at 24 hr) were inhibited by TGF-$\beta$ sRII, a TGF-$\beta$ inhibitor. These results suggest that TGF-$\beta$ is part of the cascade of events underlying long-term sensitization, and also indicate that a signaling molecule used in development may also have functions in adult neuronal plasticity. ^

Tuning Molecular Recognition of RNA and DNA via Sugar Modification

This minireview highlights three aspects of our recent work in the area of sugar modified oligonucleotide analogues. It provides an overview over recent results on the conformationally constrained analogue tricyclo-DNA with special emphasis of its antisense properties, it summarizes results on triple-helix forming oligodeoxynucleotides containing pyrrolidino-nucleosides with respect to DNA recognition via the dual recognition mode, and it highlights the advantageous application of the orthogonal oligonucleotidic pairing system homo-DNA in molecular beacons for DNA diagnostics

Dehydrin expression as a potential diagnostic tool for cold stress in white clover

Cold acclimation is important for crop survival in environments undergoing seasonal low temperatures. It involves the induction of defensive mechanisms including the accumulation of different cryoprotective molecules among which are dehydrins (DHN). Recently several sequences coding for dehydrins were identified in white clover (Trifolium repens). This work aimed to select the most responsive to cold stress DHN analogues in search for cold stress diagnostic markers. The assessment of dehydrin transcript accumulation via RT-PCR and immunodetection performed with three antibodies against the conserved K-, Y-, and S-segment allowed to outline different dehydrin types presented in the tested samples. Both analyses confirmed that YnKn dehydrins were underrepresented in the controls but exposure to low temperature specifically induced their accumulation. Strong immunosignals corresponding to 37–40 kDa with antibodies against Y- and K-segment were revealed in cold-stressed leaves. Another ‘cold-specific’ band at position 52–55 kDa was documented on membranes probed with antibodies against K-segment. Real time RT-qPCR confirmed that low temperatures induced the accumulation of SKn and YnSKn transcripts in leaves and reduced their expression in roots. Results suggest that a YnKn dehydrin transcript with GenBank ID: KC247805 and the immunosignal at 37–40 kDa, obtained with antibodies against Y- and K-segment are reliable markers for cold stress in white clover. The assessment of SKn (GenBank ID: EU846208) and YnSKn (GenBank ID: KC247804) transcript levels in leaves could serve as additional diagnostic tools.

Regression of the inflammatory microenvironment of the peritoneal cavity in women with endometriosis by GnRHa treatment.

OBJECTIVE To investigate the effect of gonadotropin-releasing hormone analogues (GnRHa) on the peritoneal fluid microenvironment in women with endometriosis. STUDY DESIGN Peritoneal fluid was collected from 85 women with severe endometriosis (rAFS stage III and IV) during laparoscopic surgery during the proliferative phase. Prior to surgery clinical data were collected. The concentrations of specific markers for endometriosis in the peritoneal fluid were determined using an ELISA and a comparison between peritoneal fluid markers in women using GnRHa and no hormonal treatment was performed using a non-parametric Mann-Whitney U test. RESULTS The study included peritoneal fluid from 39 patients who had been administered GnRHa (Zoladex(®)) in the three months prior to surgery and 46 from women with no hormonal treatment in this period. Concentrations of IL-8, PAPP-A, glycodelin-A and midkine were significantly reduced in the GnRHa treatment group compared to women receiving no hormonal treatment. RANTES, MCP-1, ENA-78, TNF-α, OPG, IP-10 and defensin showed no significant change between the two groups. CONCLUSIONS GnRHa mediate a significant regression in the inflammatory nature of the peritoneal microenvironment in women with endometriosis.

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the uptake of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter

Synthesis and Biochemical Characterization of Tricyclothymidine Triphosphate (tc-TTP)

Tricyclo-DNA (tc-DNA) is a conformationally restricted oligonucleotide analogue that exhibits promising properties as a robust antisense agent. Here we report on the synthesis and biochemical characterization of tc-TTP, the triphosphate of a tc-DNA nucleoside containing the base thymine. Tc-TTP turned out to be a substrate for the Vent (exo−) DNA polymerase, a polymerase that allows for multiple incorporations of tc-T nucleotides under primer extension reaction conditions. However, the substrate acceptance is rather low, as also observed for other sugar-modified analogues. Tc-TTP and tc-nucleotide-containing templates do not sustain enzymatic polymerization under physiological conditions; this indicates that tc-DNA-based antisense agents will not enter natural metabolic pathways that lead to long-term toxicity.

A framework for benchmarking of homogenisation algorithm performance on the global scale

The International Surface Temperature Initiative (ISTI) is striving towards substantively improving our ability to robustly understand historical land surface air temperature change at all scales. A key recently completed first step has been collating all available records into a comprehensive open access, traceable and version-controlled databank. The crucial next step is to maximise the value of the collated data through a robust international framework of benchmarking and assessment for product intercomparison and uncertainty estimation. We focus on uncertainties arising from the presence of inhomogeneities in monthly mean land surface temperature data and the varied methodological choices made by various groups in building homogeneous temperature products. The central facet of the benchmarking process is the creation of global-scale synthetic analogues to the real-world database where both the "true" series and inhomogeneities are known (a luxury the real-world data do not afford us). Hence, algorithmic strengths and weaknesses can be meaningfully quantified and conditional inferences made about the real-world climate system. Here we discuss the necessary framework for developing an international homogenisation benchmarking system on the global scale for monthly mean temperatures. The value of this framework is critically dependent upon the number of groups taking part and so we strongly advocate involvement in the benchmarking exercise from as many data analyst groups as possible to make the best use of this substantial effort.

[111In-DOTA]LTT-SS28, a first pansomatostatin radioligand for in vivo targeting of somatostatin receptor-positive tumors

Radiolabeled pansomatostatin-like analogues are expected to enhance the diagnostic sensitivity and to expand the clinical indications of currently applied sst2-specific radioligands. In this study, we present the somatostatin mimic [DOTA]LTT-SS28 {[(DOTA)Ser1,Leu8,D-Trp22,Tyr25]SS28} and its 111In radioligand. [DOTA]LTT-SS28 exhibited a pansomatostatin-like profile binding with high affinity to all five hsst1-hsst5 subtypes (IC50 values in the lower nanomolar range). Furthermore, [DOTA]LTT-SS28 behaved as an agonist at hsst2, hsst3, and hsst5, efficiently stimulating internalization of the three receptor subtypes. Radioligand [111In-DOTA]LTT-SS28 showed good stability in the mouse bloodstream. It displayed strong and specific uptake in AR42J tumors 4 h postinjection (9.3±1.6% ID/g vs 0.3±0.0% ID/g during sst2 blockade) in mice. Significant and specific uptake was also observed in HEK293-hsst2-, HEK293-hsst3-, and HEK293-hsst5-expressing tumors (4.43±1.5, 4.88±1.1, and <3% ID/g, respectively, with values of <0.5% ID/g during receptor blockade). In conclusion, the somatostatin mimic [111In-DOTA]LTT-SS28 specifically localizes in sst2-, sst3-, and sst5-expressing xenografts in mice showing promise for multi-sst1-sst5 targeted tumor imaging.

Olefinic peptide nucleic acid (OPA)

The olefinic peptide nucleic acid analogues (OPA) monomers containing the bases thymine and adenine were synthesised in 11 steps. Fully modified oligomers containing these units were prepared and their pairing properties assessed by means of UV-melting experiments

Watson-Crick base-pairing properties of tricyclo-DNA

Tricyclo-DNA belongs to the family of conformationally restricted oligodeoxynucleotide analogues. It differs structurally from DNA by an additional ethylene bridge between the centers C(3') and C(5') of the nucleosides, to which a cyclopropane unit is fused for further enhancement of structural rigidity. The synthesis of the hitherto unknown tricyclodeoxynucleosides containing the bases cytosine and guanine and of the corresponding phosphoramidite building blocks is described, as well as a structural description of a representative of an alpha- and a beta-tricyclodeoxynucleoside by X-ray analysis. Tricyclodeoxynucleoside building blocks of all four bases were used for the synthesis of fully modified mixed-base oligonucleotides. Their Watson-Crick pairing properties with complementary DNA, RNA, and with itself were investigated by UV melting curves, CD spectroscopy, and molecular modeling. Tricyclo-DNA was found to be a very stable Watson-Crick base-pairing system. A UV melting curve analysis of the decamers tcd(pcgtgacagtt) and tcd(paactgtcacg) showed increased thermal stabilities of up to DeltaT(m)/mod. = +1.2 degrees C with complementary DNA and +2.4 degrees C with complementary RNA. With itself, tricyclo-DNA showed an increase in stability of +3.1 degrees C/base pair relative to DNA. Investigations into the thermodynamic properties of these decamers revealed an entropic stabilization and an enthalpic destabilization for the tricyclo-DNA/DNA duplexes. CD spectroscopic structural investigations indicated that tricyclo-DNA containing duplexes preferrably exist in an A-conformation, a fact which is in agreement with results from molecular modeling

Synthesis and incorporation into a-DNA of a novel conformationally constrained alpha-nucleoside analogue

We describe the synthesis and incorporation into alpha-DNA of a novel conformationally constrained alpha-nucleoside analogue. The carbohydrate part of this analogue was prepared in 4 steps from the known bicyclic precursor 1 via a stereospecific, intramolecular, Et 3B mediated radical addition to a keto-function as the key step. The thus obtained intermediate 4 was transformed stereoselectively into the corresponding alpha-nucleoside analogues 7 and 8 containing the bases adenine and thymine, and were further elaborated into the phosphoramidite building blocks 11 and 12 . Both building blocks were incorporated into alpha-oligodeoxynucleotides and their pairing behavior to parallel complementary DNA studied by UV-melting experiments. Single substitutions of alpha-deoxyribnucleoside units by the new analogues in the center of duplexes were found to be thermally destabilizing by only -0.8 to -3.1›C.

Synthesis and Thermodynamic and Biophysical Properties of Tricyclo-DNA

The DNA analogue tricyclo-DNA, built from conformationally rigid nucleoside analogues that were linked via tertiary phosphodiester functions, can efficiently be synthesized from the corresponding phosphoramidites by conventional solid-phase cyanoethyl phosphoramidite chemistry. 5'-End phosphorylated tricyclo-DNA sequences are chemically stable in aqueous, pH-neutral media at temperatures from 0 to 90 C. Tricyclo-DNA sequences resist enzymatic hydrolysis by the 3'-exonuclease snake venom phosphodiesterase. Homobasic adenine- and thymine-containing tricyclo-DNA octa- and nonamers are extraordinarily stable A-T base-pairing systems, not only in their own series but also with complementary DNA and RNA. Base mismatch formation is strongly destabilized. As in bicyclo-DNA, the tricyclo-DNA purine sequences preferentially accept a complementary strand on the Hoogsteen face of the base. A thermodynamic analysis reveals entropic benefits in the case of hetero-backbone duplex formation (tricyclo-DNA/DNA duplexes) and both an enthalpic and entropic benefit for duplex formation in the pure tricyclo-DNA series compared to natural DNA. Stability of tricyclo-DNA duplex formation depends more strongly on monovalent salt concentration compared to natural DNA. Homopyrimidine DNA sequences containing tricyclothymidine residues form triplexes with complementary double-stranded DNA. Triple-helix stability depends on the sequence composition and can be higher when compared to that of natural DNA. The use of one tricyclothymidine residue in the center of the self-complementary dodecamer duplex (d(CGCGAAT t CGCG), t = tricyclothymidine) strongly stabilizes its monomolecular hairpin loop structure relative to that of the corresponding pure DNA dodecamer ( T m = +20 C), indicating (tetra)loop-stabilizing properties of this rigid nucleoside analogue.