James Guthrie

Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans.

Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1) gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.

The Cation Diffusion Facilitator Gene cdf-2 Mediates Zinc Metabolism in Caenorhabditis elegans

Zinc is essential for many cellular processes. To use Caenorhabditis elegans to study zinc metabolism, we developed culture conditions allowing full control of dietary zinc and methods to measure zinc content of animals. Dietary zinc dramatically affected growth and zinc content; wild-type worms survived from 7 μm to 1.3 mm dietary zinc, and zinc content varied 27-fold. We investigated cdf-2, which encodes a predicted zinc transporter in the cation diffusion facilitator family. cdf-2 mRNA levels were increased by high dietary zinc, suggesting cdf-2 promotes zinc homeostasis. CDF-2 protein was expressed in intestinal cells and localized to cytosolic vesicles. A cdf-2 loss-of-function mutant displayed impaired growth and reduced zinc content, indicating that CDF-2 stores zinc by transport into the lumen of vesicles. The relationships between three cdf genes, cdf-1, cdf-2, and sur-7, were analyzed in double and triple mutant animals. A cdf-1 mutant displayed increased zinc content, whereas a cdf-1 cdf-2 double mutant had intermediate zinc content, suggesting cdf-1 and cdf-2 have antagonistic functions. These studies advance C. elegans as a model of zinc metabolism and identify cdf-2 as a new gene that has a critical role in zinc storage.

ttm-1 encodes CDF transporters that excrete zinc from intestinal cells of C. elegans and act in a parallel negative feedback circuit that promotes homeostasis.

Zinc is an essential metal involved in a wide range of biological processes, and aberrant zinc metabolism is implicated in human diseases. The gastrointestinal tract of animals is a critical site of zinc metabolism that is responsible for dietary zinc uptake and distribution to the body. However, the role of the gastrointestinal tract in zinc excretion remains unclear. Zinc transporters are key regulators of zinc metabolism that mediate the movement of zinc ions across membranes. Here, we identified a comprehensive list of 14 predicted Cation Diffusion Facilitator (CDF) family zinc transporters in Caenorhabditis elegans and demonstrated that zinc is excreted from intestinal cells by one of these CDF proteins, TTM-1B. The ttm-1 locus encodes two transcripts, ttm-1a and ttm-1b, that use different transcription start sites. ttm-1b expression was induced by high levels of zinc specifically in intestinal cells, whereas ttm-1a was not induced by zinc. TTM-1B was localized to the apical plasma membrane of intestinal cells, and analyses of loss-of-function mutant animals indicated that TTM-1B promotes zinc excretion into the intestinal lumen. Zinc excretion mediated by TTM-1B contributes to zinc detoxification. These observations indicate that ttm-1 is a component of a negative feedback circuit, since high levels of cytoplasmic zinc increase ttm-1b transcript levels and TTM-1B protein functions to reduce the level of cytoplasmic zinc. We showed that TTM-1 isoforms function in tandem with CDF-2, which is also induced by high levels of cytoplasmic zinc and reduces cytoplasmic zinc levels by sequestering zinc in lysosome-related organelles. These findings define a parallel negative feedback circuit that promotes zinc homeostasis and advance the understanding of the physiological roles of the gastrointestinal tract in zinc metabolism in animals.

The DAF-16 FOXO Transcription Factor Regulates natc-1 to Modulate Stress Resistance in Caenorhabditis elegans, Linking Insulin/IGF-1 Signaling to Protein N-Terminal Acetylation

The insulin/IGF-1 signaling pathway plays a critical role in stress resistance and longevity, but the mechanisms are not fully characterized. To identify genes that mediate stress resistance, we screened for C. elegans mutants that can tolerate high levels of dietary zinc. We identified natc-1, which encodes an evolutionarily conserved subunit of the N-terminal acetyltransferase C (NAT) complex. N-terminal acetylation is a widespread modification of eukaryotic proteins; however, relatively little is known about the biological functions of NATs. We demonstrated that loss-of-function mutations in natc-1 cause resistance to a broad-spectrum of physiologic stressors, including multiple metals, heat, and oxidation. The C. elegans FOXO transcription factor DAF-16 is a critical target of the insulin/IGF-1 signaling pathway that mediates stress resistance, and DAF-16 is predicted to directly bind the natc-1 promoter. To characterize the regulation of natc-1 by DAF-16 and the function of natc-1 in insulin/IGF-1 signaling, we analyzed molecular and genetic interactions with key components of the insulin/IGF-1 pathway. natc-1 mRNA levels were repressed by DAF-16 activity, indicating natc-1 is a physiological target of DAF-16. Genetic studies suggested that natc-1 functions downstream of daf-16 to mediate stress resistance and dauer formation. Based on these findings, we hypothesize that natc-1 is directly regulated by the DAF-16 transcription factor, and natc-1 is a physiologically significant effector of the insulin/IGF-1 signaling pathway that mediates stress resistance and dauer formation. These studies identify a novel biological function for natc-1 as a modulator of stress resistance and dauer formation and define a functionally significant downstream effector of the insulin/IGF-1 signaling pathway. Protein N-terminal acetylation mediated by the NatC complex may play an evolutionarily conserved role in regulating stress resistance.

Specific activity measurement of 64Cu: A comparison of methods

Effective specific activity of (64)Cu (amount of radioactivity per µmol metal) is important in order to determine purity of a particular (64)Cu lot and to assist in optimization of the purification process. Metal impurities can affect effective specific activity and therefore it is important to have a simple method that can measure trace amounts of metals. This work shows that ion chromatography (IC) yields similar results to ICP mass spectrometry for copper, nickel and iron contaminants in (64)Cu production solutions.

Fast and reliable method for As speciation in urine samples containing low levels of As by LC-ICP-MS: Focus on epidemiological studies

The speciation analysis of As in urine samples can provide important information for epidemiological studies. Considering that these studies involve hundreds or thousands of samples, a fast and reliable method using a simple LC system with short-length mixed bed ion exchange chromatographic column coupled to ICP-MS for As speciation in human urine samples was developed in this work. Separation of AB+TMAO, DMA, AC, MMA and AsIII+AsV was accomplished within 5min with good resolution when ammonium carbonate solutions were used as mobile phases and H2O2 was added to samples to quantitatively convert AsIII-AsV. Repeatability studies yielded RSD values from 2.0% to 4.8% for all species evaluated. Limits of detection (LOD) for As species ranged from 0.003 to 0.051ngg-1. Application of the method to human urine samples from a non-contaminated area showed that the sum of species measured corresponded to 62-125% of the total As in the sample. The recovery values for these species in urine SRM 2669 were in the range of 89-112% and demonstrated the suitability of the proposed method for epidemiological studies.

Bulk production and evaluation of high specific activity 186g Re for cancer therapy using enriched 186 WO 3 targets in a proton beam

INTRODUCTION Rhenium-186g (t1/2 = 3.72 d) is a β- emitting isotope suitable for theranostic applications. Current production methods rely on reactor production by way of the reaction 185Re(n,γ)186gRe, which results in low specific activities limiting its use for cancer therapy. Production via charged particle activation of enriched 186W results in a 186gRe product with a higher specific activity, allowing it to be used more broadly for targeted radiotherapy applications. This targets the unmet clinical need for more efficient radiotherapeutics. METHODS A target consisting of highly enriched, pressed 186WO3 was irradiated with protons at the Los Alamos National Laboratory Isotope Production Facility (LANL-IPF) to evaluate 186gRe product yield and quality. LANL-IPF was operated in a dedicated nominal 40 MeV mode. Alkaline dissolution followed by anion exchange chromatography was used to isolate 186gRe from the target material. Phantom and radiolabeling studies were conducted with the produced 186gRe activity. RESULTS A 186gRe batch yield of 1.38 ± 0.09 MBq/μAh or 384.9 ± 27.3 MBq/C was obtained after 16.5 h in a 205 μA average/230μA maximum current proton beam. The chemical recovery yield was 93% and radiolabeling was achieved with efficiencies ranging from 60-80%. True specific activity of 186gRe at EOB was determined via ICP-AES and amounted to 0.788 ± 0.089 GBq/μg (0.146 ± 0.017 GBq/nmol), which is approximately seven times higher than the product obtained from neutron capture in a reactor. Phantom studies show similar imaging quality to the gold standard 99mTc. CONCLUSIONS We report a preliminary study of the large-scale production and novel anion exchange based chemical recovery of high specific activity 186gRe from enriched 186WO3 targets in a high-intensity proton beam with exceptional chemical recovery and radiochemical purity.

Gelatinase activity imaged by activatable cell-penetrating peptides in cell-based and in vivo models of stroke.

Matrix metalloproteinases (MMPs), particularly gelatinases (MMP-2/-9), are involved in neurovascular impairment after stroke. Detection of gelatinase activity in vivo can provide insight into blood–brain barrier disruption, hemorrhage, and nerve cell injury or death. We applied gelatinase-activatable cell-penetrating peptides (ACPP) with a cleavable l-amino acid linker to examine gelatinase activity in primary neurons in culture and ischemic mouse brain in vivo. We found uptake of Cy5-conjugated ACPP (ACPP-Cy5) due to gelatinase activation both in cultured neurons exposed to n -methyl-d-aspartate and in mice after cerebral ischemia. Fluorescence intensity was significantly reduced when cells or mice were treated with MMP inhibitors or when a cleavage-resistant ACPP-Cy5 was substituted. We also applied an ACPP dendrimer (ACPPD) conjugated with multiple Cy5 and/or gadolinium moieties for fluorescence and magnetic resonance imaging (MRI) in intact animals. Fluorescence analysis showed that ACPPD was detected in sub-femtomole range in ischemic tissues. Moreover, MRI and inductively coupled plasma mass spectrometry revealed that ACPPD produced quantitative measures of gelatinase activity in the ischemic region. The resulting spatial pattern of gelatinase activity and neurodegeneration were very similar. We conclude that ACPPs are capable of tracing spatiotemporal gelatinase activity in vivo, and will therefore be useful in elucidating mechanisms of gelatinase-mediated neurodegeneration after stroke.

Development of ammonium bifluoride fusion method for rapid dissolution of trinitite samples and analysis by ICP-MS

Field deployable dissolution techniques are needed to decrease response time following a nuclear event. This study tested the capability of NH4HF2 fusion to dissolve trinitite. Dissolution of trinitite by NH4HF2 fusion was compared to conventional microwave digestion. Following digestion, trace elements were measured using ICP-MS. This work demonstrates that low temperature fusion with NH4HF2 is capable of dissolving trinitite samples. The low purity of the NH4HF2 resulted in a higher limit of detection for several elements when compared to microwave digestion with high purity acids.

The use of 111Ag as a tool for studying biological distribution of silver-based antimicrobials

Recently, there has been an emergence of significant interest in silver-based antimicrobials. Our goal was to develop a radioactive tracer for investigating the biological fate of such compounds. Purified 111Ag was incorporated into the methylated caffeine analogue, IC1 to yield the silver carbene complex designated as [111Ag]SCC1 and investigated in biodistribution studies.