Altered [99mTc]Tc-MDP biodistribution from neutron activation sourced 99Mo.

Given potential worldwide shortages of fission sourced 99Mo/99mTc medical isotopes there is increasing interest in alternate production strategies. A neutron activated 99Mo source was utilized in a single center phase III open label study comparing 99mTc, as 99mTc Methylene Diphosphonate ([99mTc]Tc-MDP), obtained from solvent generator separation of neutron activation produced 99Mo, versus nuclear reactor produced 99Mo (e.g., fission sourced) in oncology patients for which an [99mTc]Tc-MDP bone scan would normally have been indicated. Despite the investigational [99mTc]Tc-MDP passing all standard, and above standard of care, quality assurance tests, which would normally be sufficient to allow human administration, there was altered biodistribution which could lead to erroneous clinical interpretation. The cause of the altered biodistribution remains unknown and requires further research.

Reduced catabolic protein expression in Clostridium butyricum DSM 10702 correlate with reduced 1,3-propanediol synthesis at high glycerol loading.

Higher initial glycerol loadings (620 mM) have a negative effect on growth and 1,3-propanediol (1,3-PDO) synthesis in Clostridium butyricum DSM 10702 relative to lower initial glycerol concentrations (170 mM). To help understand metabolic shifts associated with elevated glycerol, protein expression levels were quantified by LC/MS/MS analyses. Thirty one (31) proteins involved in conversion of glycerol to 1,3-PDO and other by-products were analyzed by multiple reaction monitoring (MRM). The analyses revealed that high glycerol concentrations reduced cell growth. The expression levels of most proteins in glycerol catabolism pathways were down-regulated, consistent with the slower growth rates observed. However, at high initial glycerol concentrations, some of the proteins involved in the butyrate synthesis pathways such as a putative ethanol dehydrogenase (CBY_3753) and a 3-hydroxybutyryl-CoA dehydrogenase (CBY_3045) were up-regulated in both exponential and stationary growth phases. Expression levels of proteins (CBY_0500, CBY_0501 and CBY_0502) involved in the reductive pathway of glycerol to 1,3-PDO were consistent with glycerol consumption and product concentrations observed during fermentation at both glycerol concentrations, and the molar yields of 1,3-PDO were similar in both cultures. This is the first report that correlates expression levels of glycerol catabolism enzymes with synthesis of 1,3-PDO in C. butyricum. The results revealed that significant differences in the expression of a small subset of proteins were observed between exponential and stationary growth phases at both low and high glycerol concentrations.

Unifying expression scale for peptide hydrophobicity in proteomic reversed phase high-pressure liquid chromatography experiments.

As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.

Functional activity of murine intestinal mucosal cells is regulated by the glucagon-like peptide-1 receptor.

To determine whether the glucagon-like peptide-1 receptor (GLP-1r) plays a role in the regulation of intestinal functional activity, we analyzed the distribution of the GLP-1r in mouse tissues and tested if tissues expressing the receptor respond to exendin-4 and exendin (9-39) amide, a GLP-1r agonist and antagonist respectively. In ileum, Glp1r mRNA level was two fold higher in extracts from epithelial cells than non-epithelial tissues. By immunohistochemistry, the receptor was localized to the mucosal cell layer of villi of ileum and colon, to the myenteric and submucosal plexus and to Paneth cells. Intravenous administration of exendin-4 to CD-1 mice induced expression of the immediate early gene c-fos in mucosal cells but not in cells of the enteric plexuses or in L cells of ileum. The induction of c-fos was inhibited by the voltage-gated sodium channel blocker tetrodotoxin. Exendin-4 also increased c-fos expression in ileal segments in vitro, suggesting that this action of the analog was independent of an extrinsic input. The induction of c-fos expression by exendin-4 was inhibited by exendin (9-39) amide, indicating that the action of exendin-4 was mediated by activation of the receptor. Our findings indicate that the GLP-1r is involved in ileal enterocyte and Paneth cell function, that the GLP-1 analog activates c-fos expression in the absence of an extrinsic input and that some of the actions of the receptor is/are mediated by voltage-gated Na channels.

Phenotype of entero-endocrine L cells becomes restricted during development.

BACKGROUND: Glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP) are hormones secreted by L and K cells, respectively, and by LK cells. To characterize L and K cells during development, we examined ileum from embryonic (e)- 12 to e-17. RESULTS: GLP-1 cells were first seen at e-15 and their number increased at e-17. At e-17, most GLP-1 cells co-expressed GIP. The transcription factors Pax6 and Pdx-1 are required for GIP expression, while Pax6 activates the expression of GLP-1. At e-17, the mucosa has GIP+ Pax6+, GIP+ Pdx-1+, GLP-1+ Pax6+, and GLP-1+ Pdx-1+ cells. Unlike ileal L cells of postnatal and adult mice, a subset of ileal L cells of e-17 embryos co-expressed GLP-1 and glucagon (Glu). Glu-positive cells contain proprotein-convertase 2 (PC2) and PC3/1, the enzymes responsible for Glu and GLP-1 synthesis, respectively. CONCLUSIONS: Our findings indicate that most GLP-1+ cells of ileum of e-17 embryos co-express GIP and, therefore, are LK cells. In addition, a subset of GLP-1+ cells of embryos but not of neonates co-express glucagon, indicating that the expression of Glu in GLP-1+ cells disappears after birth.

Regulation of mouse intestinal L cell progenitors proliferation by the glucagon family of peptides.

Glucagon like peptide-1 (GLP-1) and GLP-2 are hormones secreted by intestinal L cells that stimulate glucose-dependent insulin secretion and regulate intestinal growth, respectively. Mice with deletion of the glucagon receptor (Gcgr) have high levels of circulating GLP-1 and GLP-2. We sought to determine whether the increased level of the glucagon-like peptides is due to L cell hyperplasia. We found, first, that high levels of the glucagon-like peptides increase L cell number but does not affect the number of other intestinal epithelial cell types. Second, a large proportion of ileal L cells of Gcgr(-/-) mice coexpressed glucose-dependent insulinotropic peptide (GIP). Cells coexpressing GIP and GLP-1 are termed LK cells. Third, the augmentation in L cell number was due to a higher rate of proliferation of L cell progenitors rather than to the entrance of mature L cells into the cell cycle. Fourth, a high concentration of the glucagon-like peptides in the circulation augmented the mRNA levels of transcription factors expressed by late but not early enteroendocrine progenitors. Fifth, the administration of exendin 9-39, a GLP-1 receptor antagonist, resulted in a decrease in the rate of L cell precursor proliferation. Finally, we determined that L cells do not express the GLP-1 receptor, suggesting that the effect of GLP-1 is mediated by paracrine and/or neuronal signals. Our results suggest that GLP-1 plays an important role in the regulation of L cell number.

Predicting retention time shifts associated with variation of the gradient slope in peptide RP-HPLC.

We have developed a sequence-specific model for predicting slopes (S) in the fundamental equation of linear solvent strength theory for the reversed-phase HPLC separation of tryptic peptides detected in a typical bottom-up-proteomics experiment. These slopes control the variation in the separation selectivity observed when the physical parameters of chromatographic separation, such as gradient slope, flow rate, and column size are altered. For example, with the use of an arbitrarily chosen set of tryptic peptides with a 4-times difference in the gradient slope between two experiments, the R(2)-value of correlation between the observed retention times of identical species decreases to ~0.993-0.996 (compared to a theoretical value of ~1.00). The observed retention time shifts associated with variations of the gradient slope can be predicted a priori using the approach described here. The proposed model is based on our findings for a set of synthetic species (Vu, H.; Spicer, V.; Gotfrid, A.; Krokhin, O. V. J. Chromatogr., A, 2010, 1217, 489-497), which postulate that slopes S can be predicted taking into account simultaneously peptide length, charge, and hydrophobicity. Here we extend this approach using an extensive set of real tryptic peptides. We developed the procedure to accurately measure S-values in nano-RP HPLC MS experiments and introduced sequence-specific corrections for a more accurate prediction of the slopes S. A correlation of ~0.95 R(2)-value between the predicted and experimental S-values was demonstrated. Predicting S-values and calculating the expected retention time shifts when the physical parameters of separation like gradient slope are altered will facilitate a more accurate application of peptide retention prediction protocols, aid in the transfer of scheduled MRM (SRM) procedures between LC systems, and increase the efficiency of interlaboratory data collection and comparison.

Differential expression of glucagon and glucagon-like peptide 1 receptors in mouse pancreatic alpha and beta cells in two models of alpha cell hyperplasia.

Glucose homeostasis is determined by a balance between insulin and glucagon, produced by beta and alpha cells of the pancreas respectively. The levels of circulating hormones is partly determined by the mass of these two endocrine cell types. However, in contrast to beta cells, the identity of the signals regulating alpha cell number is not known. Mice with a global deletion of the glucagon receptor (Gcgr-/-) and mice with ablation of prohormone convertase 2 (PC2), the enzyme involved in the conversion of proglucagon into mature glucagon, develop alpha cell hyperplasia. These observations and the fact that Gcgr-/- mice exhibit high levels of circulating glucagon-like peptide-1 (GLP-1) suggested that members of the glucagon family of peptides could be directly involved in the regulation of alpha cell number. In this study we sought to determine whether alpha cells express receptors for Glucagon (Gcgr) and/or the glucagon-like peptide-1 (GLP1r). We examined the expression of these receptors in islets of Gcgr-/-, PC2-/- mice and control littermates, in an alpha (alphaTC1/9) and in a beta (betaTC3) cell line. Gcgr was expressed exclusively by islet beta cells, but not by alpha cells, of the two lines of mice lacking glucagon signaling. Similarly, betaTC but not alphaTC cells, expressed Gcgr. The expression of GLP1r by alpha cells was determined by the genotype and age of the mice. In embryos, GLU+ cells of Gcgr+/+ mice cells express GLP1r during early development, but not in adults. In contrast, alpha cells of Gcgr-/- mice were GLP1r+ throughout life, reflecting the immature state of GLU+ cells when Gcgr is deleted. Unlike alpha cells, beta cells of all mice lines examined initiate GLP1r expression after birth. These results suggest that GLP-1 may affect the maturation of postnatal but not prenatal beta cells. In addition, they also suggest that the incretin could mediate alpha cell proliferation, inducing the development of alpha cell hyperplasia in Gcgr-/- mice.

Mass spectral characterization of organophosphate-labeled lysine in peptides.

Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.

Mass spectrometry identifies multiple organophosphorylated sites on tubulin.

Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 degrees C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.