Labeling proteins using aryl iodide acylation agents: Influence of meta vs para substitution on in vivo stability

The effect of para vs meta substitution on the biological behavior of an intact antibody and an F(ab′)₂ fragment was investigated. Paired-label studies were performed using 81C6 IgG and OC 125 F(ab′)₂ labeled using the N-succinimidyl esters of both p-[¹²⁵I]- and m-[¹³¹I]iodobenzoate as well as with the potential catabolites, p-[¹²⁵I]- and m-[¹³¹I]iodobenzoic acid. In all 3 studies, up to 55% lower uptake of ¹³¹I in thyroid and stomach was observed, suggesting that the m-substituted species were more inert to dehalogenation in vivo.     

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [¹²⁵I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [¹²⁵I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [¹²⁵I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[¹²⁵I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [¹²⁵I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[¹²⁵I]Fab to free polysomes was also obtained. The [¹²⁵I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[¹²⁵I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.     

Labeling a TCO-functionalized single domain antibody fragment with 18F via inverse electron demand Diels Alder cycloaddition using a fluoronicotinyl moiety-bearing tetrazine derivative

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with ¹⁸F (t_½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for ¹⁸F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel’s Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [¹⁸F]AlF-NOTA moiety with [¹⁸F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[¹⁸F]fluoronicotinyl-PEG₄-methyltetrazine to provide [¹⁸F]FN-PEG₄-Tz-TCO-GK-PEG₄-5F7 ([¹⁸F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[¹²⁵I]iodobenzoate (iso-[¹²⁵I]SGMIB) also were synthesized. Radiochemical purity, affinity (K_D) and immunoreactive fraction of [¹⁸F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [¹⁸F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[¹²⁵I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [¹⁸F]FN-GK-5F7 were 3–4 times higher than those for co-injected iso-[¹²⁵I]SGMIB-5F7 as well as those observed for the ¹⁸F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [¹⁸F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [¹⁸F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem.     

Extracellular matrix metabolism by chondrocytes 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

THe incorporation of [³H]glycine into acid-insoluble protein and of [³H]acetate into glysoaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gardients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of poly-ribosomes that was reversed by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence on L-glutamine was also demonstrated for other avain connective tissues.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Comparative Affinities of Adrenomedullin (AM) and Calcitonin Gene-Related Peptide (CGRP) for [125I] AM and [125I] CGRP Specific Binding Sites in Porcine Tissues

Abstract

We have investigated the binding characteristics of rat [¹²⁵I] adrenomedullin (AM) and human [¹²⁵I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [¹²⁵I] rat AM and [¹²⁵I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [¹²⁵I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [¹²⁵I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [¹²⁵I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [¹²⁵I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

Detection of a 65 kDaras binding protein in rat and sheep brain cytosol using a chemical cross linking agent

The ability of aras protein to associate with proteins present in rat brain cytosolin vitro was investigated using chemical cross-linking agents and the¹²⁵I-labelled v-H-ras protein. Two iodinated protein complexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [¹²⁵I]ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-PAGE. Formation of the [¹²⁵I] 85 kDa complex was enhanced by a high concentration of EDTA while generation of the 40 kDa species was abolished by this treatment. Formation of the [¹²⁵I] 85 kDa complex was inhibited by unlabelledras protein, GTP, GTPS, and GDP but not by ATPS and GMP.Chromatography of the cross-linked brain cytosol-[¹²⁵I]ras mixture on DEAE cellulose partially resolved the [¹²⁵I] 85 kDa complex from the [¹²⁵I]ras protein. The [¹²⁵I] 85 kDa complex (formed using ethyleneglycolbis (succinimidylsuccinate) as the cross-linking agent) could be immunoprecipitated using a rabbit anti-ras polyclonal antibody. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [¹²⁵I]-labelledras. A substantial enrichment of the proportion of the [¹²⁵I] 85 kDa complex in the cross-linked extract was achieved by preparative SDS-PAGE. It is concluded that thein vitro chemical cross-linking approach employed here has detected tworas binding proteins in rat brain cytosol: a 65 kDa heat-sensitive and a 20 kDa heat-stable protein. The possibility that the 65 kDaras binding protein is aras regulatory orras effector protein which has not so far been characterised is briefly discussed.Abbreviations DSS disuccinimidyl suberate - EGS ethyleneglycolbis (succinimidylsuccinate) - GTPS guanosine 5-[-thio] triphosphate - ATPS adenosine 5-[-thio] triphosphate     

Immunohistochemical localization of articular cartilage proteoglycan and link protein in situ using monoclonal antibodies and lectin-binding methods

Lectins have specificity for certain carbohydrate structures in macromolecules. Lectins are, therefore, useful histochemical tools for demonstrating the composition and localization of components of connective tissue matrices, such as articular cartilage. In order to assess the significance of observed lectin-binding patterns, experiments were performed in which monoclonal antibodies against chondroitin sulphate- and keratan sulphate-containing proteolgycans and link proteins were applied to sections of bovine articular cartilage after enzymatic digestion with chondroitinase ABC and keratanase. The following conclusions were made: (1) Binding of peanut agglutinin (PNA) in the interterritorial matrix predominantly indicates the presence of keratan sulphate, but may also detectO-linked oligosaccharides of proteoglycans. (2) In normal cartilage wheat germ agglutinin (WGA) binds nearly exclusively to keratan sulphate. In cartilage degraded with chondroitinase ABC and keratanase this lectin may also detect carbohydrates in link protein due to enhanced accessibility. Binding of WGA toO-linked oligosaccharides may eventually occur. (3) In enzymatically digested cartilage matrix, staining with soybean agglutinin (SBA) may be due to link protein, but not to chondroitin sulphate, because specific breakdown of the glycosaminoglycan chain is required for binding of SBA. (4)Ulex europaeus agglutinin I (UEA I) binding sites are only detectable in digested cartilage matrix.     

Pharmacokinetics and lung distribution of a humanized anti-RAGE antibody in wild-type and RAGE-/- mice

A neutralizing antibody to the receptor for the advanced glycation end products (anti-RAGE Ab) was developed as a potential treatment of acute and chronic inflammatory conditions. Previous pharmacology studies demonstrated efficacy of the anti-RAGE antibody in the mouse model of sepsis. We examined pharmacokinetics and lung distribution of [¹²⁵I]anti-RAGE Ab in RAGE-/- and wild-type (129S5) mice following single IV administration. Serum pharmacokinetics of [¹²⁵I]anti-RAGE Ab was similar in RAGE-/- and 129S5 mice, with the total body clearance of 0.3 mL/hr/kg and the elimination half-life of 11-12 days, suggesting the target expression had limited impact on overall elimination of [¹²⁵I]anti-RAGE Ab from mice. [¹²⁵I]Anti-RAGE Ab accumulated in the lung of 129S5 mice, with ~4% of total dose retained in the lung at days 6-27 and the lung AUC0-∞ of ~300% of that in serum. The SDS-PAGE analysis suggested that most of retained lung radioactivity was attributed to intact antibody. No accumulation of radioactivity was observed in the lung of RAGE-/- mice, indicating that lung uptake of [¹²⁵I]anti-RAGE Ab was target-dependent in wild-type mice. These data suggest that the anti-RAGE Ab was able to localize to the site of RAGE expression, the lung, and support the findings in the previous pharmacology studies.     

Possibility for tumor detection with fatty acid analogs

Newly synthesized 17-[¹⁸F]fluoro-3-methylheptadecanoic acid ([¹⁸F]BMHDA), 16-[¹⁸F]fluoropalmitic acid ([¹⁸F]PA) and 15-(p-[¹²⁵I]iodophenyl)-3-R,S-methylpentadecanoic acid ([¹²⁵I]BMIPP), fatty acid tracers, were examined for the possibility of tumor imaging using 10 tumor models in rats and mice. The highest tumor/muscle ratios with [¹⁸F]BMHDA were 2.3 using rat tumor AH109A and 1.9 using mouse tumor B16F1 (tumor accumulation 0.41 ± 0.05, 4.29 ± 0.77% ID/g, respectively). Tumor/muscle ratios with [¹²⁵I]BMIPP and [¹⁸F]PA were lower than those with [¹⁸F]BMHDA. Labeled fatty acids seem to have a lower potential for tumor detection.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

The Mechano-Ubiquitinome of Articular Cartilage: Differential Ubiquitination and Activation of a Group of ER-Associated DUBs and ER Stress Regulators

Understanding how connective tissue cells respond to mechanical stimulation is important to human health and disease processes in musculoskeletal diseases. Injury to articular cartilage is a key risk factor in predisposition to tissue damage and degenerative osteoarthritis. Recently, we have discovered that mechanical injury to connective tissues including murine and porcine articular cartilage causes a significant increase in lysine-63 polyubiquitination. Here, we identified the ubiquitin signature that is unique to injured articular cartilage tissue upon mechanical injury (the “mechano-ubiquitinome”). A total of 463 ubiquitinated peptides were identified, with an enrichment of ubiquitinated peptides of proteins involved in protein processing in the endoplasmic reticulum (ER), also known as the ER-associated degradation response, including YOD1, BRCC3, ATXN3, and USP5 as well as the ER stress regulators, RAD23B, VCP/p97, and Ubiquilin 1. Enrichment of these proteins suggested an injury-induced ER stress response and, for instance, ER stress markers DDIT3/CHOP and BIP/GRP78 were upregulated following cartilage injury on the protein and gene expression levels. Similar ER stress induction was also observed in response to tail fin injury in zebrafish larvae, suggesting a generic response to tissue injury. Furthermore, a rapid increase in global DUB activity following injury and significant activity in human osteoarthritic cartilage was observed using DUB-specific activity probes. Combined, these results implicate the involvement of ubiquitination events and activation of a set of DUBs and ER stress regulators in cellular responses to cartilage tissue injury and in osteoarthritic cartilage tissues. This link through the ER-associated degradation pathway makes this protein set attractive for further investigation in in vivo models of tissue injury and for targeting in osteoarthritis and related musculoskeletal diseases.     

Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.     

Assessment of [125I]WYE-230949 as a Novel Histamine H3 Receptor Radiopharmaceutical

Histamine H₃ receptor therapeutics have been proposed for several diseases such as schizophrenia, attention deficit hyperactivity disorder, Alzheimer''s disease and obesity. We set out to evaluate the novel compound, [¹²⁵I]WYE-230949, as a potential radionuclide imaging agent for the histamine H₃ receptor in brain. [¹²⁵I]WYE-230949 had a high in vitro affinity for the rat histamine H₃ receptor (K_d of 6.9 nM). The regional distribution of [¹²⁵I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H₃ receptor. Rat brain uptake of intravenously injected [¹²⁵I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [¹²⁵I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [¹²⁵I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [¹²⁵I]WYE230949 is not a potential radiotracer for imaging rat histamine H₃ receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.     

[125I]MIBG uptake and release in different regions of the rat brain

Radioiodinated m-iodobenzylguanidine ([¹²⁵I]MIBG) and tritiated norepinephrine ([³H]NE]) uptake and release were compared, in different regions of the brain of the rat. The classification of the regions according to uptake was the same for both tracers: striatum > hypothalamus > hippocampus > cortex > brainstem. Tetrabenazine (TBZ), a granular monoamine uptake inhibitor reduced the uptake in the different regions. The inhibition rate was higher for [³H]NE uptake than for [¹²⁵I]MIBG. The spontaneous release was the same for [¹²⁵I]MIBG and [³H]NE and was the lowest in the striatum. The K⁺ stimulated release of [³H]NE was more complete than the release of [¹²⁵I]MIBG and was the most important in the striatum. From these results, it is inferred that MIBG enters the brain tissue via NE uptake mechanisms. It appears that MIBG is stored in the chromaffin granules, as NE, but also in the cytoplasm. A modified molecule derived from MIBG which would cross the blood-brain barrier, would then appear as a potential scintigraphic marker of monoamine uptake, storage and release.     

Diesterified Derivatives of 5-Iodo-2′-Deoxyuridine as Cerebral Tumor Tracers

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2′-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[¹²⁵I]iodo-3′,5′-di-O-acetyl-2′-deoxyuridine (Ac₂[¹²⁵I]IUdR), 5-[¹²⁵I]iodo-3′,5′-di-O-pivaloyl-2′-deoxyuridine (Piv₂[¹²⁵I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac₂[¹²⁵I]IUdR, Piv₂[¹²⁵I]IUdR and [¹²⁵I]IUdR (control) were prepared with labeling yields of 31–47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [¹²⁵I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv₂[¹²⁵I]IUdR>Ac₂[¹²⁵I]IUdR>[¹²⁵I]IUdR. For Ac₂[¹²⁵I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [¹²⁵I]IUdR, Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability.     

Reversed-phase high-performance liquid chromatography of salmon calcitonin and its degradation products in biological samples using column switching and flow-through radioisotope detection

For the determination of salmon calcitonin and its degradation products in biological samples, a reversed-phase HPLC method with column switching and flow-through radioisotope detection has been developed using high specific activity [¹²⁵I]salmon calcitonin. Effects of the precolumn packing material and washing solvent were examined in terms of [¹²⁵I]salmon calcitonin recovery. Spiked samples of [¹²⁵I]salmon calcitonin in plasma and kidney homogenate were injected onto a LiChroprep RP-8 precolumn after dilution with 0.1% trifluoroacetic acid. After washing the polar interfering compounds with 0.1% trifluoroacetic acid, the concentrated [¹²⁵I]salmon calcitonin and its degradation products were eluted and separated on a W-Porex C₁₈ column with a gradient of 0.1% trifluoroacetic acid in acetonitrile-water. Detection and calibration of [¹²⁵I]salmon calcitonin were possible down to picogram levels. Reproducible kinetic data for the degradation of intact [¹²⁵I]salmon calcitonin by rat kidney homogenate could be traced.     

Identification of link proteins in canine synovial cell cultures and canine articular cartilage

Link proteins are glycoproteins in cartilage that are involved in the stabilization of aggregates of proteoglycans and hyaluronic acid. We have identified link proteins in synovial cell cultures form normal canine synovium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunofluorescence, and immunolocation with specific antibodies by electrophoretic transfer. We have also found evidence for the synthesis of link proteins in these cultures by fluorography of radiolabeled synovial cell extracts. We have identified a 70,000 mol-wt protein in canine synovial cell culture extracts that has antigenic cross-reactivity with the 48,000-mol-wt link protein. Three link proteins were identified in normal canine articular cartilage. These results indicate that link proteins are more widely distributed in connective tissues than previously recognized and may have biological functions other than aggregate stabilization.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.