Linker modification reduced the renal uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to examine the biodistribution of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH in B16/F1 melanoma-bearing C57 mice to determine whether the replacement of the Lys linker with an Arg linker could decrease the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH. ^99mTc-RAD-Arg-(Arg¹¹)CCMSH exhibited rapid and high tumor uptake (17.98 ± 4.96% ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice. As compared to ^99mTc-RAD-Lys-(Arg¹¹)CCMSH, the replacement of the Lys linker with an Arg linker dramatically decreased the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH by 68%, 62%, 73% and 64% at 0.5, 2, 4 and 24 h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2 h post-injection using ^99mTc-RAD-Arg-(Arg¹¹)CCMSH as an imaging probe.     

Tc-99m-labeled RGD-conjugated alpha-melanocyte stimulating hormone hybrid peptides with reduced renal uptake

The purpose of this study was to examine whether the replacement of the positively-charged Lys or Arg linker with a neutral linker could reduce the renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys^3,4,10, D-Phe⁷, Arg¹¹]α-MSH_3-13 {(Arg¹¹)CCMSH} through the neutral βAla or Ahx {aminohexanoic acid} linker (replacing the Lys or Arg linker) to generate novel RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH hybrid peptides. The receptor-binding affinity and cytotoxicity of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH were determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH and ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The replacement of the Lys or Arg linker with the βAla or Ahx linker retained nanomolar receptor-binding affinities and remarkable cytotoxicity of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH. The receptor-binding affinities of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH were 0.8?±?0.05 and 1.3?±?0.1 nM. Three-hour incubation with 0.1 µM of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH decreased the survival percentages of B16/F1 cells by 71 and 67 % as compared to the untreated control cells 5 days post the treatment. The replacement of the Arg linker with the βAla or Ahx linker reduced the non-specific renal uptake of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH and ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH by 62 and 61 % at 2 h post-injection. ^99mTc-RGD-βAla-(Arg¹¹)CCMSH displayed higher melanoma uptake than ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH at 0.5, 2, 4, and 24 h post-injection. Enhanced tumor to kidney uptake ratio of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH warranted the further evaluation of ¹⁸⁸Re-labeled RGD-βAla-(Arg¹¹)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.     

Substitution of Gly with Ala enhanced the melanoma uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to determine the melanoma targeting property of (99m)Tc-RAD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice and compare with (99m)Tc-RGD-Lys-(Arg(11))CCMSH we previously reported. (99m)Tc-RAD-Lys-(Arg(11))CCMSH exhibited rapid and high tumor uptake (19.91±4.02% ID/g at 2h post-injection) in B16/F1 melanoma-bearing C57 mice. The tumor uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH was 1.51, 1.34 and 1.43 times the tumor uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH at 0.5, 2 and 4h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2h post-injection using (99m)Tc-RAD-Lys-(Arg(11))CCMSH as an imaging probe. The substitution of Gly with Ala significantly enhanced the melanoma uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH compared to (99m)Tc-RGD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice, providing a new insight into the design of α-MSH peptides for melanoma targeting.     

Synthesis and biological evaluation of a novel 99mTc nitrido radiopharmaceutical with deoxyglucose dithiocarbamate,showing tumor uptake

The deoxyglucose dithiocarbamate (DGDTC) was synthesized and radiolabelled with [^99mTcN]²⁺ intermediate to form the ^99mTcN–DGDTC complex. The radiochemical purity of the ^99mTcN–DGDTC complex was over 90%, as measured by TLC and by HPLC, without any notable decomposition at room temperature over a period of 6 h. The partition coefficient and electrophoresis results indicated that this complex was hydrophilic and neutral. The biodistribution of ^99mTcN–DGDTC in mice bearing S 180 tumor showed that the complex accumulated in the tumor with high uptake and good retention. The tumor/blood and tumor/muscle ratios increased with time and reached 2.32 and 1.68 at 4 h post-injection, suggesting it would be a promising candidate for tumor imaging.     

A novel concept of radiosynthesis of a 99mTc-labeled dimeric RGD peptide as a potential radiotracer for tumor imaging

Radiolabeled Arg-Gly-Asp (RGD) peptides are promising agents for non invasive imaging of α_vβ₃ expression in malignant tumors. The integrin α_vβ₃ binding affinity and consequent tumor uptake could be improved when a dimeric RGD peptide is used as the targeting moiety instead of a monomer. Towards this, a novel approach was envisaged to synthesize a ^99mTc labeled dimeric RGD derivative using a RGD monomer and [^99mTcN]⁺² intermediate. The dithiocarbamate derivative of cyclic RGD peptide G₃-c(RGDfK) (G₃ = Gly-Gly-Gly, f = Phe, K = Lys) was synthesized and radiolabeled with [^99mTcN]⁺² intermediate to form the ^99mTcN-[G₃-c(RGDfK)]₂ complex in high yield (～98%). Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed good tumor uptake [4.61 ± 0.04% IA/g at 30 min post-injection] with fast clearance of the activity from non-target organs/tissue. Scintigraphic imaging studies showed visible accumulation of activity in the tumor with appreciable target to background ratio.     

Synthesis and evaluation of 99mTc–2-[(3-carboxy-1-oxopropyl)amino]-2-deoxy-d-glucose as a potential tumor imaging agent

The 2-[(3-carboxy-1-oxopropyl)amino]-2-deoxy-d-glucose (CPADG) was synthesized and radiolabeled with ^99mTcO₄⁻ to obtain the ^99mTc–CPADG complex in high yield. It was stable over 6 h in saline at room temperature and in serum at 37 °C. The partition coefficient and electrophoresis results indicated that the complex was hydrophilic and cationic. In vitro cell studies showed there was an increase in the uptake of ^99mTc–CPADG as a function of incubation time and ^99mTc–CPADG was possibly transported via the glucose transporters. The biodistribution of ^99mTc–CPADG in mice bearing S 180 tumor showed that the complex accumulated in the tumor with high uptake and good retention. The tumor/blood and tumor/muscle ratios increased with time and reached 1.91 and 5.05 at 4 h post-injection. Single photon emission computed tomography (SPECT) image studies showed there was an obvious accumulation in tumor sites, suggesting ^99mTc–CPADG would be a promising candidate for tumor imaging.     

Preparation of classical Re/99mTc(CO)3+ and novel 99mTc(CO)2(NO)2+ cores complexed with flavonol derivatives and their binding characteristics for Aβ(1–40) aggregates

Classical ^99mTc(CO)₃⁺ and novel ^99mTc(CO)₂(NO)²⁺ cores complexed with flavonol derivatives were prepared. Autoradiography of postmortem AD transgenic mice (Tg C57, APP, PS1 12-month-old) brain section confirmed the binding property of [^99mTc(CO)₃⁺-3-OH-flavone]⁰ to Aβ_(1–40) aggregates, while the novel ^99mTc(CO)₂(NO)²⁺ labeled compounds showed no binding sites in AD transgenic mice sections. Intravenous administration of [^99mTc(CO)₃⁺-3-OH-flavone]⁰ resulted in moderate brain uptake (0.48 ± 0.05%ID/g) at 5 min post-injection and slow clearance from the brain issues in 2 h post-injection (120 min: 0.39 ± 0.08%ID/g). Then an Aβ_(1–40)-receptor-targeted Re(CO)₃⁺-3-OH-flavone, was prepared to identify the structure of the technetium complex. UV–vis absorption and fluorescence emission properties have been studied at room temperature in order to determine the natures of the lowest electronically excited states of Re(CO)₃⁺-3-OH-flavone and the ligand. The fluorescent rhenium complex Re(CO)₃⁺-3-OH-flavone showed high affinity for Aβ_(1–40) aggregates in vitro by fluorescence spectra (dissociation constant (K_d) = 11.16 nM). In conclusion, the results suggested that ^99mTc(CO)₃⁺-3-OH-flavone should be a suitable candidate as Aβ plaque SPECT imaging agent for AD.     

Synthesis and biological evaluation of a novel 99mTc cyclopentadienyl tricarbonyl complex ([(Cp-R)99mTc(CO)3]) for sigma-2 receptor tumor imaging

We report the design, synthesis and biological evaluation of a novel ^99mTc 4-(4-cyclohexylpiperazine-1-yl)-butan-1-one-1-cyclopentadienyltricarbonyl technetium ([^99mTc]5) as a potential SPECT tracer for imaging of σ₂ receptors in tumors. [^99mTc]5 was prepared in 25 ± 5% isolated radiochemical yield with radiochemical purity of >99% via double-ligand transfer (DLT) reaction from the ferrocene precursor 2b (4-(4-cyclohexylpiperazine-1-yl)-1-ferrocenylbutan-1-one). The corresponding Re-complex 4 and the ferrocenyl complex 2b showed relatively high affinity towards σ₂ receptors in in vitro competition binding assay (K_i values of 4 and 2b were 64.4 ± 18.5 nM and 43.6 ± 21.3 nM, respectively) and moderate to high selectivity versus σ₁ receptors (K_iσ₁/K_iσ₂ ratios were 12.5 and 95.5, respectively). The log D value of [^99mTc]5 was determined to be 2.52 ± 0.33. Biodistribution studies in mice revealed comparably high initial brain uptake of [^99mTc]5 and slow washout. Administration of haloperidol 5 min prior to injection of [^99mTc]5 significantly reduced the radiotracer uptake in brain, heart, lung, and spleen by 40–50% at 2 h p.i.. Moreover, [^99mTc]5 showed high uptake in C6 glioma cell lines (8.6%) after incubation for 1 h. Blocking with haloperidol to compete with [^99mTc]5 significantly reduced the cell uptake. Preliminary blocking study in C6-brain-tumor bearing rats showed that [^99mTc]5 binds to σ receptors in the brain-tumor specifically. These results are encouraging for further exploration of ^99mTc-labeled probes for σ₂ receptor tumor imaging in vivo.     

Synthesis and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel ^99mTc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC₅₀ values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC₅₀ = 4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({^99mTc(CO)₃}⁺) to afford the {^99mTc(CO)₃}⁺ complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {^99mTc(CO)₃}⁺ radiolabeled PSMA inhibitors.     

Evaluation of 99mTc-probestin for imaging APN expressing tumors by SPECT

Aminopeptidase N (APN) is known to play important roles in tumor angiogenesis, tumor cell invasion, and metastasis. Thus, APN is an attractive biomarker for imaging tumor angiogenesis. Here we report results obtained from biodistribution and single photon emission computed tomography (SPECT) imaging studies of a technetium-99m labeled probestin (a potent APN inhibitor) conjugate containing a tripeptide, Asp-DAP-Cys (DAP = 2,3-diaminopropionic acid), chelator and a 8-amino-3,6-dioxaoctanoic acid (PEG₂) linker conducted in nude mice xenografted with HT-1080 human fibrosarcoma tumors (APN-positive tumors). These results collectively demonstrate that ^99mTc-probestin uptake by tumors and other APN expressing tissues in vivo is specific and validate the use of probestin as a vector for targeting APN in vivo.     

Melanoma targeting property of a Lu-177-labeled lactam bridge-cyclized alpha-MSH peptide

The purpose of this study was to determine the melanoma targeting property of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing C57 mice. ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex exhibited high receptor-mediated melanoma uptake and fast urinary clearance. The tumor uptake of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex was 20.25 ± 4.59 and 21.63 ± 6.27% ID/g at 0.5 and 2 h post-injection, respectively. Approximately 83% of injected dose cleared out the body via urinary system at 2 h post-injection. ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex were 2.76 and 1.74 at 2 and 24 h post-injection. The melanoma lesions were clearly visualized by SPECT/CT using ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection. Overall, high melanoma uptake coupled with fast urinary clearance of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex underscored its potential for melanoma treatment in the future.     

Novel cancer-targeting SPECT/NIRF dual-modality imaging probe 99mTc-PC-1007: Synthesis and biological evaluation

Synthesis, characterization, in vitro and in vivo biological evaluation of a heptamethine cyanine based dual-mode single-photon emission computed tomography (SPECT)/near infrared fluorescence (NIRF) imaging probe ^99mTc-PC-1007 is described. ^99mTc-PC-1007 exhibited preferential accumulation in human breast cancer MCF-7 cells. Cancer-specific SPECT/CT and NIRF imaging of ^99mTc-PC-1007 was performed in a breast cancer xenograft model. The probe uptake ratio of tumor to control (spinal cord) was calculated to be 4.02 ± 0.56 at 6 h post injection (pi) and 8.50 ± 1.41 at 20 h pi (P < 0.0001). Pharmacokinetic parameters such as blood clearance and organ distribution were assessed.     

Inorganic and organic nitrogen uptake by the toxigenic diatom Pseudo-nitzschia australis (Bacillariophyceae)

The nitrogen uptake capabilities of the toxigenic diatom Pseudo-nitzschia australis (Frenguelli), freshly isolated from Monterey Bay California, were examined in unialgal laboratory cultures at saturating photosynthetic photon flux densities (100 μmol photons m⁻² s⁻¹) and 15 °C. The kinetics of nitrogen (nitrate, ammonium, urea and glutamine) uptake as a function of substrate concentration were estimated from short (20.5 min) incubations using the ¹⁵N-tracer technique. Based on the estimated maximum specific uptake rates and measures of N affinity (the initial slope of the uptake versus nutrient concentration curve), nitrate is the preferred nitrogen substrate, followed by glutamine and ammonium, which are equivalent. Rates of urea uptake by P. australis did not saturate at concentrations as high as 36 μg-at N L⁻¹, and urea uptake as a function of concentration could not be described by Michaelis–Menten kinetics over the concentration gradient tested. Although there is a clear preference for nitrate at equivalent concentrations (compared to ammonium, urea, and glutamine), these laboratory results demonstrate the capability of this pennate diatom to utilize both inorganic and organic forms of nitrogen, supporting field observations that P. australis blooms during both upwelling and non-upwelling conditions off the west coast of North America. Substantial differences in the nitrogenous nutrition of P. australis can be expected in these environments, and anthropogenic inputs of N substrates such as ammonium and urea can support its growth, and may contribute significantly to both harmful diatom blooms and the maintenance of seed populations at non-bloom abundances, particularly during periods of reduced or absent upwelling.     

99mTc-tricarbonyl labeled agents for cell labeling: Development,biodistribution in normal mice and preliminary in vitro evaluation

We have developed four ^99mTc(CO)₃-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. ^99mTc(CO)₃-hexadecylamino-N,N′-diacetic acid (negatively charged), ^99mTc(CO)₃-hexadecylamino-N-α-picolyl-N′-acetic acid (uncharged), ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine (positively charged), ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with ^99mTc-d,l-HMPAO and [¹⁸F]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine after 60 min incubation (compared to 55% and 23% for ^99mTc-d,l-HMPAO and [¹⁸F]FDG, respectively) while in plasma-rich medium, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine was best bound (54%, similar to the binding of ^99mTc-d,l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent (^99mTc(CO)₃-N,N′-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent ^99mTc-d,l-HMPAO, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Chara hispida beds as a sink of nitrogen: Evidence from growth,nitrogen uptake and decomposition

Chara hispida forms dense beds (0.78–0.95 kg DW m⁻²) in Colgada Lake. The ability of Chara meadows to act as a nitrogen source or sink was evaluated by the following methods: (1) investigating Chara growth, (2) nitrogen incorporation and decomposition laboratory experiments and (3) relating experimental results to field conditions. Sediment oospores were germinated in large aquaria and observed growth rates were 0.001 m day⁻¹ (shoot length) and 0.0002 g day⁻¹ (dry weight). Nitrogen uptake rates were determined by addition of K¹⁵NO₃ during two different periods of Chara growth and the rates were 1.21 and 3.86 μM g DW⁻¹ h⁻¹ when charophytes were 166 days old (not sexually mature) and 323 days old (sexually mature), respectively. After the uptake experiments, the same charophytes were allowed to decompose within two types of litter bags (3 mm-pore litter bags and entire, non-porous plastic litter bags). Decomposition rates of Ch. hispida were 0.016 and 0.009 day⁻¹ in perforated and non-perforated bags, respectively, and fit a negative exponential model. The nitrogen release rate, calculated as the disappearance of N content from Chara tissues, was 0.012 day⁻¹ and there were no statistically significant differences between the values from the two different bag types. The dissolved organic nitrogen concentrations in aquarium and non-perforated litter bags waters increased linearly with time due to the leaching of soluble compounds from Chara. The rate of N loss from Chara tissues, total nitrogen and dissolved organic nitrogen release rates and the decrease in initial dry weight rate were all lower than the daily rate of Chara N uptake. By extrapolating laboratory data to field situations, we determined that approximately 38% of the N taken up by charophytes in Colgada Lake during the growth period is retained. Given the high charophyte biomass in the lake, its ability to incorporate nitrogen, its low decomposition rate and its ability to over-winter, we conclude that Chara beds could be acting as nitrogen sinks in this ecosystem.     

Short and long-term uptake of Hg in white lupin plants:Kinetics and stress indicators

The intrinsic characteristics of white lupin regarding biomass production and tolerance to abiotic stresses could make it a good candidate to be used in degraded mine soils containing mercury (Hg), but white lupin behaviour in response to Hg has to be previously evaluated. With this aim, kinetic parameters of Hg uptake in short and long-term experiments, and Hg resistance of white lupin plants using several stress indicators were studied. The plants were grown with increasing Hg doses in nutrient solutions (0, 5 and 10 μM). Hg uptake showed an active component in Hg influx, suggesting the existence of a low affinity root transporter that can be used for Hg uptake into white lupin root cells. K_m and V_max values obtained for the saturable component were 217.7 ± 27.6 μM and 3.78 ± 0.18 μmol Hg g FW^?1 h^?1. Hg accumulation was concentration–time-dependent, showing a saturable behaviour for the lower doses but a linear behaviour for the highest ones. A high ability of Hg absorption by white lupin was observed both in short and long-term uptake experiments. The highest Hg dose reduced biomass production especially in the shoots. Moreover, increases in chlorophylls, malondialdehyde, total thiols and phenols were observed in Hg-stressed plants. The enhancement of total thiols and phenols levels in roots reduced oxidative stress for the 5 μM dose, but not for higher Hg levels. The deleterious effect of Hg was less marked in root tissues, in spite of their accumulation of very high Hg amounts (99%) because of, at least in part, a combined increase in total thiols and phenolics able to minimize oxidative stress. Our results suggested that phenolic content in roots could be used as a new and easy-to-use indicator of Hg presence. On the whole, white lupin showed a certain ability to survive in Hg-contaminated media and it would be possible to include it in combined decontamination strategies.     

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12–14) to c-Met receptor positive U87MG cells.     

Synthesis and characterization of 123I-CMICE-013: A potential SPECT myocardial perfusion imaging agent

Coronary artery disease (CAD) is a major cause of death in Canada and the United States. Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is a useful diagnostic test in the management of patients with CAD. The widely used SPECT MPI agents, ^99mTc sestamibi and ^99mTc tetrofosmin, exhibit less than ideal pharmacokinetic properties with decreasing uptake with higher flows. ¹²³I has a similar energy as ^99mTc, an ideal half life, and is readily available from cyclotrons. The objective of this study was to develop an ¹²³I labeled MPI agent based on rotenone, a mitochondrial complex I inhibitor, as an alternative to currently available SPECT MPI agents. Methods: ¹²³I-CMICE-013 was synthesized by radiolabeling rotenone with ¹²³I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, followed by RP-HPLC purification. The product was formulated in 5% EtOH in 10 mM NaOAc pH 6.5. The inactive analog ¹²⁷I-CMICE-013 was isolated and characterized by NMR and mass spectrometry, and the structure determined. Micro-SPECT imaging studies were carried out in normal and infarcted rats. Biodistribution studies were performed in normal rats at 2 h (n = 6) and 24 h (n = 8) post injection (p.i.). Results: ¹²³I-CMICE-013 was isolated with >95% radiochemical purity and high specific activity (14.8–111 GBq/μmol; 400–3000 mCi/μmol). Structural analysis showed that rotenone was iodinated at 7′-position, with removal of the 6′,7′-double bond, and addition of a hydroxy group at 6′-position. MicroSPECT images in normal rats demonstrated homogeneous and sustained myocardial uptake with minimal interference from lung and liver. Absent myocardial perfusion was clearly identified in rats with permanent left coronary artery ligation and ischemia-reperfusion injury. In vivo biodistribution studies in normal rats at 2 h p.i. showed significant myocardial uptake (2.01 ± 0.48%ID/g) and high heart to liver (2.98 ± 0.93), heart to lung (4.11 ± 1.04) and heart to blood (8.37 ± 3.97) ratios. At 24 h p.i., the majority of ¹²³I-CMICE-013 was cleared from tissues, and a significant amount of tracer was found in the thyroid, indicating in vivo deiodination of the tracer. Conclusion: ¹²³I-CMICE-013 is a promising new radiotracer for SPECT MPI with high myocardial uptake, very good target to background ratios and favorable biodistribution characteristics.     

Foliage type specific susceptibility to ozone in Picea abies,Pinus cembra and Larix decidua at treeline: A synthesis

Cumulative ozone uptake (COU, mmol m⁻²) and O₃ flux (FO₃, nmol m⁻² s⁻¹) were related to physiological, morphological and biochemical characteristics of field-grown mature evergreen Norway spruce [Picea abies (L.) Karst.], Cembran pine [Pinus cembra L.], and deciduous European larch [Larix decidua Mill.] trees at treeline. The threshold COU causing a statistically significant decline in photosynthetic capacity (A_max) ranged between 19.6 mmol m⁻² in current-year needles of evergreen conifers and 22.0 6 mmol m⁻² in short-shoot needles of deciduous L. decidua subjected to exposure periods of ≥84 and ≥43 days, respectively. The higher O₃ sensitivity of deciduous L. decidua than of evergreen P abies and P. cembra was associated with differences in FO₃ and specific leaf area (SLA), both being significantly higher in L. decidua. FO₃ was 5.9 nmol m⁻² s⁻¹ in L. decidua and 2.7 nmol m⁻² s⁻¹ in evergreen conifers. Species-dependent differences were also related to detoxification capacity expressed through total surface area based concentrations of reduced ascorbate and α-tocopherol that both increased with SLA. Findings suggest that differences in O₃ sensitivity between evergreen and deciduous conifers can be attributed to foliage type specific differences in SLA, the latter determining physiological and biochemical characteristics of the treeline conifers.