Synthesis and characterization of two (111)In-labeled DTPA-peptide conjugates.

This report describes the synthesis and characterization of two (111)In-labeled DTPA-peptide conjugates (DTPA-MA and DTPA-BA). It is surprising to find that (111)In(DTPA-MA) and (111)In(DTPA-BA) are more hydrophilic than their corresponding (90)Y analogues, suggesting a different coordination sphere in (111)In and(90)Y complexes of the same DTPA-peptide conjugate. By a reversed phase HPLC method, both (111)In(DTPA-MA) and (111)In(DTPA-BA) showed only one radiometric peak in their respective HPLC chromatogram due to a rapid interconversion of different isomers (particularly cis and trans isomers for (111)In(DTPA-MA); cis-cis, cis-trans, trans-cis, and trans-trans isomers for (111)In(DTPA-BA)). The interconversion of different isomers involves the "wagging" of the diethylenetriamine backbone, "shuffling" of the NO or NO(2) donor sets, and a rapid inversion at the terminal amine-nitrogen atoms.     

99mTc-labeling of a hydrazinonicotinamide-conjugated vitronectin receptor antagonist useful for imaging tumors.

This report describes the (99m)Tc labeling of a HYNIC-conjugated vitronectin receptor antagonist (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo[Lys-Arg-Gly-Asp-D-Phe])-cyclo[Lys-Arg-Gly-Asp-D-Phe]). The ternary ligand complex [(99m)Tc(SQ168)(tricine)(TPPTS)] (RP593) was prepared using a non-SnCl(2)-containing formulation. The corresponding (99)Tc analogue, [(99)Tc]RP593, was also prepared and characterized by HPLC and LC-MS. A HPLC concordance experiment using RP593 and [(99)Tc]RP593 showed that the same technetium complex was prepared at both the tracer and macroscopic levels. The LC-MS data is completely consistent with the 1:1:1:1 composition for Tc:SQ168:tricine:TPPTS and provides direct evidence that the two radiometric peaks in the radio-HPLC chromatogram of RP593 are indeed due to the resolution of diastereomers. In an in vitro receptor binding assay, [(99)Tc]RP593 was shown to have comparable binding affinity for the vitronectin receptor to that of SQ168 itself.     

Synthesis and evaluation of two technetium-99m-labeled peptidic 2-nitroimidazoles for imaging hypoxia.

The presence of hypoxic cells in solid tumors is a marker for therapy-resistant, aggressive disease. The noninvasive detection of hypoxic cells in tumors by radiolabeled 2-nitroimidazoles is a diagnostic technique under current evaluation. Two peptidic agents, dimethylglycyl-L-seryl-L-cysteinyl-lysyl{N(epsilon)-[1-(2-nitro-1H -im idazolyl)acetamido]}glycine (RP435) and dimethylglycyl-tert-butylglycyl-L-cysteinyl-glycine-[2-(2-ni tro-1H-im idazolyl)ethyl]amide (RP535) have been synthesized. Both agents contain an N(3)S class chelator for (99m)Tc and Re and a 2-nitroimidazole group which can be enzymatically reduced and selectively trapped in cells under hypoxic conditions. Two isomers of (99m)TcO-RP435, which are assumed to be syn and anti conformations, were observed on HPLC analysis. The interconversion of the two isomers in aqueous solution was investigated. In contrast, RP535 chelated (99m)Tc to form a single isomer and no conversion to its counterpart has been observed on HPLC analysis. The tert-butyl group on the chelator may inhibit the formation and interconversion of the syn and anti isomers of (99m)TcO-RP535. Both tracers showed a significant degree of hypoxia-specific accumulation in an in vitro assay, with (99m)TcO-RP535 showing higher selectivity for hypoxic cells than (99m)TcO-RP435. These results suggest that (99m)TcO-RP535 represents a lead compound worthy of further investigation as an agent for imaging hypoxia in tumors.     

Preparation and characterization of paramagnetic polychelates and their protein conjugates

The gadolinium complexes of poly-L-lysine-poly(diethylenetriamine-N,N,N',N",N"-pentaacetic acid) (Gd-PL-DTPA) and poly-L-lysine-poly(1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetr aacetic acid) (Gd-PL-DOTA) and their conjugates with human serum albumin (HSA) have been prepared and characterized. Poly-L-lysine (PL, degree of polymerization approximately 100) was N-acylated with a mixed anhydride of the chelating ligand (DTPA or DOTA). Sixty to ninety chelating groups per molecule of PL could be attached in this way. Following purification of the polychelate by size-exclusion chromatography, the gadolinium complexes were prepared by standard methods and conjugated to HSA with heterobifunctional cross-linking reagents. The molar relaxities of these macromolecular species were 2-3-fold higher than those of the corresponding monomeric metal complexes [( Gd(DTPA)] and [Gd(DOTA)]). The conjugation conditions were optimized to produce conjugates containing 60-90 metal centers per molecule of HSA (ca. one polychelate per protein).     

High-level conjugation of chelating agents onto immunoglobulins: use of an intermediary poly(L-lysine)-diethylenetriaminepentaacetic acid carrier

Diethylenetriaminepentaacetic acid (DTPA), a strong chelating agent, was covalently linked to murine monoclonal anti-HLA IgG1 antibody (H-1) with the use of poly(L-lysine) (Mr 14,000) as a multivalent, intermediary carrier, via thiol-disulfide exchange reaction. The conjugates contained up to 42.5 mol DTPA per mol antibody, and retained over 90% of their antibody activity in vitro. The conjugates incorporated gadolinium (Gd) through an exchange reaction with Gd-EDTA, used to prevent colloid formation and nonspecific binding of the free metal. The IgG-poly(L-lysine)-DTPA-Gd had a greater effect per mol on proton relaxation rates than DTPA-Gd itself. Use of poly(L-lysine) as an intermediary carrier for attachment of chelating agents to IgG thus offers great potential for achieving high-specific-activity conjugates, particularly for use as biologically specific contrast agents in nuclear magnetic resonance imaging.     

Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides.

The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1-3/2-4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3:2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable (1)H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L-alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D (1)H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists.     

Effect of coligands on biodistribution characteristics of ternary ligand 99mTc complexes of a HYNIC-conjugated cyclic RGDfK dimer

This report describes biodistribution characteristics of three ternary ligand complexes [(99m)Tc(SQ168)(tricine)(L)] (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-d-Phe})-cyclo{Lys-Arg-Gly-Asp-d-Phe}; L = TPPTS (trisodium triphenylphosphine-3,3',3' '-trisulfonate), ISONIC (isonicotinic acid) and PDA (2,5-pyridinedicarboxylic acid)) in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts. Ternary ligand complexes [(99m)Tc(SQ168)(tricine)(L)] (L = TPPTS, ISONIC and PDA) were prepared and were analyzed by a reversed HPLC method. Surprisingly, coligands have little impact on log P values of their ternary ligand (99m)Tc complexes even though HPLC retention times suggest that [(99m)Tc(SQ168)(tricine)(PDA)] and [(99m)Tc(SQ168)(tricine)(ISONIC)] are more hydrophilic than [(99m)Tc(SQ168)(tricine)(TPPTS)]. The results from biodistribution studies indicated that excretion kinetics of the (99m)Tc-labeled cyclic RGDfK dimer can be modified by the choice of coligand. The fact that all three radiotracers show high tumor uptake during the 2 h study period suggests that the coligand has minimal effect on the tumor targeting capability of the (99m)Tc-labeled cyclic RGDfK dimer. Results from the blocking experiment suggest that the tumor localization of the (99m)Tc-labeled cyclic RGDfK dimer is integrin alpha(v)beta(3)-mediated. On the basis of their liver uptake and tumor/liver ratios, we believe that PDA has the advantage over TPPTS and ISONIC for the (99m)Tc-labeling of HYNIC-biomolecule conjugates.     

(90)Y and (177)Lu labeling of a DOTA-conjugated vitronectin receptor antagonist useful for tumor therapy.

The (90)Y and (177)Lu complexes (RP697 and RP688, respectively) of a DOTA-conjugated vitronectin receptor antagonist (SU015: 2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecyl)acetyl-Glu(cyclo[Lys-Arg-Gly-Asp-D-Phe])-cyclo[Lys-Arg-Gly-Asp-D-Phe]) were prepared by reacting SU015 with the radiometal chloride in ammonium acetate buffer (pH > 7.2) in the presence of an antioxidant (sodium gentisate, GA). Through a series of radiolabeling experiments, it was found that there are many factors influencing the rate of (90)Y chelation and the radiolabeling efficiency of SU015. These include the purity of SU015, the pH, reaction temperature, and heating time, as well as the presence of trace metal contaminants, such as Ca(2+), Fe(3+), and Zn(2+). The chelation of (90)Y by SU015 is slow, so that heating at elevated temperatures (50-100 degrees C) is needed to complete the (90)Y-labeling. The rate of (90)Y chelation is also dependent on the pH of the reaction mixture. Under optimized radiolabeling conditions (pH 7.2-7.8 and heating at 50-100 degrees C for 5-10 min), the minimum amount of SU015 required to achieve 95% RCP for RP697 is approximately 25 microg for 20 mCi of (90)YCl(3) corresponding to a SU015:(90)Y ratio of approximately 30:1.     

Proteolytic cleavage of the precursor of von Willebrand factor is not essential for multimer formation

Monkey kidney cells (COS-1), transfected with full-length human von Willebrand factor (vWF) cDNA encoding the precursor of vWF (pro-vWF), mimic the characteristics of the biosynthesis and of the constitutive secretory pathway, displayed by cultured vascular endothelial cells. Such heterologous transfected cells are able to cleave pro-vWF, generating the propolypeptide and mature vWF, and to assemble pro-vWF dimers into a series of multimers, similarly to endothelial cells. Evidence is presented showing that proteolytic processing of pro-vWF by COS-1 cells occurs at the peptide bond between arginine and serine in the sequence Lys762-Arg763-Ser764, identical to endothelial cell-associated proteolysis. This conclusion stems from the observation that substitution of Arg763 by a glycine residue completely abolishes proteolytic processing. As a result, transfection of COS-1 with the mutant vWF-Gly763 cDNA does not significantly affect the multimeric organization of secreted vWF molecules. Consequently, we conclude that proteolytic processing of pro-vWF is not required for multimer formation. Pulse-chase labeling of COS-1 cells transfected with full-length vWF cDNA reveals pro-vWF exclusively in cell lysates, whereas both pro-vWF and mature vWF are encountered in the conditioned medium. These observations indicate that proteolytic processing of pro-vWF is a "late" event during intracellular routing of these molecules or may occur extracellularly.     

Stabilization of (90)y-labeled DOTA-biomolecule conjugates using gentisic acid and ascorbic acid.

Radiolytic degradation of radiolabeled compounds is a major challenge for the development of new therapeutic radiopharmaceuticals. The goal of this study is to explore the factors influencing the solution stability of a (90)Y-labeled DOTA-peptide conjugate (RP697), including the amount of total activity, the activity concentration, the stabilizer concentration, and the storage temperature. In general, the rate of radiolytic decomposition of RP697 is much slower at the lower activity concentration (<4 mCi/mL) than that at the higher concentration (>10 mCi/mL). RP697 remains relatively stable at the 20 mCi level and room temperature while it decomposes rapidly at the 100 mCi level under the same storage conditions. Radical scavengers, such as gentisic acid (GA) and ascorbic acid (AA), were used in combination with the low temperature (-78 degrees C) to prevent the radiolytic decomposition of RP697. It was found that RP697 remains stable for at least 2 half-lives of (90)Y when GA or AA (10 mg for 20 mCi of (90)Y) is used as a stabilizer when the radiopharmaceutical composition is stored at -78 degrees C. The stabilizer (GA and AA) can be added into the formulation either before or after radiolabeling. The post-labeling approach is particularly useful when the use of a large amount of the stabilizer interferes with the radiolabeling. The radiopharmaceutical composition developed in this study can also apply to other (90)Y-labeled DOTA-biomolecule conjugates. The amount of the stabilizer used in the radiopharmaceutical composition and storage temperature should be adjusted according to the sensitivity of the radiolabeled DOTA-biomolecule conjugate toward radiolytic decomposition.     

B-Domain deleted recombinant coagulation factor VIII modified with monomethoxy polyethylene glycol

Recombinant coagulation factor VIII (r-VIII SQ) was chemically modified with monomethoxy poly(ethylene glycol) (mPEG). Three mPEG derivatives were used for coupling to the r-VIII SQ lysines, a mixed anhydride of monomethoxy poly(ethylene glycol) succinic acid (mPEG-SAH), monomethoxy poly(ethylene glycol) succinimidyl succinate (mPEG-SS), and monomethoxy poly(ethylene glycol) tresylate (mPEG-TRES). A consequence of the modification with all derivatives was a substantial reduction in coagulant activity, even at very low degrees of modification. A method was developed with the purpose of avoiding conjugation at certain important biological sites on the factor VIII and thereby producing conjugates with better retained activity. This was achieved by immobilizing the protein onto a solid matrix during the modification reaction. Characterization of conjugates by SDS-PAGE, western blots, interaction with von Willebrand factor (vWf), and thrombin activation/inactivation analyses was undertaken. The SDS-PAGE and western blots revealed coupling heterogeneity regarding degree of modification. The amount of factor VIII able to bind to vWf decreased with the conjugation. Thrombin activated the modified factor VIII to essentially the same extent as the reference preparation of r-VIII SQ. Inactivation of the modified factor VIII was, however, slower than inactivation of the unmodified protein. Finally, an in vitro study was performed to evaluate the influence of the mPEG modification on the protein stability in extract of porcine tissue. Despite that conjugates with low degrees of modification were included in the study, the coagulant activity was preserved to a significantly higher extent in all incubation mixtures containing conjugates compared to that with unmodified protein.     

Chlorophyll-a analogues conjugated with aminobenzyl-DTPA as potential bifunctional agents for magnetic resonance imaging and photodynamic therapy

A clinically relevant photosensitizer, 3-devinyl-3-(1-hexyloxyethyl)pyropheophorbide-a (HPPH, a chlorophyll-a derivative), was conjugated with Gd(III)-aminobenzyl-diethylenetriaminepentaacetic acid (DTPA), an experimental magnetic resonance (MR) imaging agent. In vivo reflectance spectroscopy confirmed tumor uptake of HPPH-aminobenzyl-Gd(III)-DTPA conjugate was higher than free HPPH administered intraveneously (iv) to C3H mice with subcutaneously (sc) implanted radiation-induced fibrosarcoma (RIF) tumor cells. In other experiments, Sprague-Dawley (SD) rats with sc implanted Ward Colon Carcinoma cells yielded markedly increased MR signal intensities from tumor regions-of-interest (ROIs) 24 h post-iv injection of HPPH-aminobenzyl-Gd(III)-DTPA conjugate as compared to unconjugated HPPH. In both in vitro (RIF tumor cells) and in vivo (mice bearing RIF tumors and rats bearing Ward Colon tumors) the conjugate produced significant increases in tumor conspicuity at 1.5 T and retained therapeutic efficacy following PDT. Also synthesized were a series of novel bifunctional agents containing two Gd(III) atoms per HPPH molecule that remained tumor-avid and PDT-active and yielded improved MR tumor conspicuity compared to their corresponding mono-Gd(III) analogues. Administered iv at a MR imaging dose of 10 micromol/kg, these conjugates produced severe skin phototoxicity. However, by replacing the hexyl group of the pyropheophorbide-a with a tri(ethylene glycol) monomethyl ether (PEG-methyl ether), these conjugates produced remarkable MR tumor enhancement at 8 h post-iv injection, significant tumoricidal activity (80% of mice were tumor-free on day 90), and reduced skin phototoxicity compared to their corresponding hexyl ether analogues. The poor water-solubility characteristic of these conjugates was resolved by incorporation into a liposomal formulation. This paper presents the synthesis of tumor-avid contrast enhancing agents for MR imaging and thus represents an important milestone toward improving cancer diagnosis and tumor characterization. More importantly, this paper describes a new family of bifunctional agents that combine two modalities into a single cost-effective "see and treat" approach, namely, a single agent that can be used for contrast agent-enhanced MR imaging followed by targeted photodynamic therapy.     

Ion-trap tandem mass spectrometric analysis of squalene monohydroperoxide isomers in sunlight-exposed human skin

We previously discovered that squalene monohydroperoxide (SQ-OOH) was produced on human forehead skin and suggested that skin squalene (SQ) may be the principal target lipid for oxidative stress (e.g., sunlight exposure). Because of its six double bonds, SQ peroxidation can yield various positional hydroperoxide isomers. However, the structural characterization of skin SQ-OOH isomers has never been reported. Here, we prepared pure SQ-OOH isomers and developed an analytical method for SQ-OOH isomers using a quadrupole/linear ion-trap mass spectrometer (QTRAP) MS/MS system. Collision-induced dissociation produced specific fragment ions for each SQ-OOH isomer, which permitted discrimination between SQ-OOH isomers by multiple reaction monitoring (MRM). When lipid extract from human forehead skin was subjected to LC-MS/MS with MRM, individual SQ-OOH isomers could be separated and detected with a sensitivity of 0.05 ng/injection. The total concentration of SQ-OOH isomers in forehead skin was approximately 956 microg/g skin lipids, but it increased up to 2,760 microg/g skin lipids after 3 h of sunlight exposure. The LC-MS/MS method was useful for investigating the peroxidation mechanisms of SQ as well as SQ-OOH-mediated skin disorders.     

Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli.

The AdhE protein of Escherichia coli is a homopolymer of 96-kDa subunits harboring three Fe(2+)-dependent catalytic functions: acetaldehyde-CoA dehydrogenase, alcohol dehydrogenase, and pyruvate formatelyase (PFL) deactivase. By negative staining electron microscopy, we determined a helical assembly of 20-60 subunits into rods of 45-120 nm in length. The subunit packing is widened along the helix axis when Fe2+ and NAD are present. Chymotrypsin dissects the AdhE polypeptide between Phe762 and Ser763, thereby retaining the alcohol dehydrogenase activity on the NH2-terminal core, but destroying all other activities. PFL deactivation, i.e. quenching of the glycyl radical in PFL by the AdhE protein, was examined with respect to cofactor involvements (Fe2+, NAD, and CoA). This process is coupled to NAD reduction and requires the intact CoA sulfhydryl group. Pyruvate and NADH are inhibitors that affect the steady-state level of the radical form of PFL in a reconstituted interconversion cycle. Studies of cell cultures found that PFL deactivation in situ is initiated at redox potentials of greater than or equal to +100 mV. Our results provide insights into the structure/function organization of the AdhE multienzyme and give a rationale for how its PFL radical quenching activity may be suppressed in situ to enable effective glucose fermentation.     

In vitro effects of cycloheximide and luteinizing hormone on the estradiol-to-progesterone shift in follicular steroidogenesis

The objectives of this study were to establish a completely in vitro system that would simulate the in vivo effects of cycloheximide (cyclo) on preovulatory serum levels of estradiol (E2) (prolonged) and progesterone (P4) (reduced). Graafian follicles were removed from proestrous hamsters at 0900 h and incubated for a basal hour (Hour 1) with various doses of cyclo before the medium was replaced; in Hour 2, 100 ng luteinizing hormone (LH) was added with cyclo added every hour for 5 or 6 h. The endpoints were steroid levels/follicle/h per ml medium of P4, 17 alpha-hydroxyprogesterone (170HP), androstenedione (A), and E2. The goal was best accomplished with hourly addition of 400 ng cyclo, which reduced follicular protein synthesis by 76%. Cyclo suppressed P4 and 170HP and prolonged the accumulation of A and E2, in Hour 5 and Hour 6, correlated with sustained thecal C-17,20-lyase/17 alpha-hydroxylase as determined by enzyme assays. Cyclo therefore prevented the early demise of the enzyme complex after LH stimulation and hence prolonged the ability of the theca to provide androgens for conversion to E2 by the granulosa cells. Our earlier work established that one of the major effects of LH is to recruit the granulosa compartment as a source of C-21 steroids, and cyclo interferes with the availability of cholesterol to mitochondrial side-chain cleavage (Greenwald and Limback, 1984). Thus, cyclo affects follicular steroidogenesis through different mechanisms in theca and granulosa.     

Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIalpha by selective mutagenesis

A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase IIalpha. Three non- conserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase IIalpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase IIalpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones.     

Role of IAA conjugates in inducing ethylene production by tobacco leaf discs

Indole-3-acetic acid (IAA) labeled in its carboxyl group was metabolized by tobacco leaf discs (Nicotiana tabacum L. cv. Xanthi) into three metabolites, two of which were preliminarily characterized as a peptide and an ester-conjugated IAA. Reapplication of each of the three metabolites (at 10 M) resulted in a marked stimulation of ethylene production and decarboxylation by the leaf discs. Similarly, these three IAA metab olites could induce elongation of wheat coleoptile segments, which was accompanied by decarboxylation. Both the exogenously supplied esteric and peptidic IAA conjugates were converted by the leaf discs into the same metabolites as free IAA. (1-¹⁴C)IAA, applied to an isolated epidermis tissue, was completely metabolized to the esteric and peptidic IAA conjugates. This epidermis tissue showed much higher ethylene production rates and lower decarboxylation rates than did the whole leaf disc.The results suggest that the participation of IAA conjugates in the regulation of various physiological processes depends on the release of free IAA, which is obtained by enzymatic hydrolysis of the conjugates in the tissue. The present study demonstrates biological activity of endogenous IAA conjugates that were synthesized by tobacco leaf discs in response to exogenously supplied IAA.Contribution No. 952-E, 1983 series, from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel.     

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.     

Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme.

In order to determine important bases at two single-stranded regions [SSrA (726-731 nt) and SSrB (762-766)] derived mainly from secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several point mutants at these two regions on the HDV88 molecule (683-770). Among the bases at SSrA and SSrB regions C763 was found to play an essential role during self-cleavage process since substitutions to any other bases viz. A or G or U completely abolished the activity.     

Stereospecific determination,chiral inversion in vitro and pharmacokinetics in humans of the enantiomers of thalidomide

The purposes of this work were (1) to develop a high performance liquid chromatographic (HPLC) assay for the enantiomers of thalidomide in blood, (2) to study their inversion and degradation in human blood, and (3) to study the pharmacokinetics of (+)-(R)- and (?)-(S)-thalidomide after oral administration of the separate enantiomers or of the racemate to healthy male volunteers. The enantiomers of thalidomide were determined by direct resolution on a tribenzoyl cellulose column. Mean rate constants of chiral inversion of (+)-(R)-thalidomide and (?)-(S)-thalidomide in blood at 37°C were 0.30 and 0.31 h^?1, respectively. Rate constants of degradation were 0.17 and 0.18 h^?1. There was rapid interconversion in vivo in humans, the (+)-(R)-enantiomer predominating at equilibrium. The pharmacokinetics of (+)-(R)- and (?)-(S)-thalidomide could be characterized by means of two one-compartment models connected by rate constants for chiral inversion. Mean rate constants for in vivo inversion were 0.17 h^?1 (R to S) and 0.12 h^?1 (S to R) and for elimination 0.079 h^?1 (R) and 0.24 h^?1 (S), i.e., a considerably faster rate of elimination of the (?)-(S)-enantiomer. Putative differences in therapeutic or adverse effects between (+)-(R)- and (?)-(S)-thalidomide would to a large extent be abolished by rapid interconversion in vivo. © 1995 Wiley-Liss, Inc.