Targeting radiopharmaceuticals: Comparative biodistribution studies of gallium and indium complexes of multidentate ligands

New multidentate ligands with structures similar to N,N′-bis[2-hydroxybenzyl]ethylenediamme-N,N′-diacetic acid (HBED) and N,N′-bis(pyridoxyl)ethylenediamine-N,N′-diacetic acid (PLED) were synthesized. The in vitro lipophilicity, electrophoretic behavior, and the in vivo biodistribution were studied for the ¹¹¹In- and ^67,68Ga-complexes of N,N′-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine-N,N′-diacetic acid (Me₄HBED); N,N′-bis(5-t-butyl-2-hydroxy-3-methylbenzyl)ethylenediamine-N,N′-diacetic acid (t-butyl HBED); N,N′-bis[2-hydroxy-5-sulfobenzyl)ethylenediamine-N,N′-diacetic acid (SHBED); N,N′-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine-N-(2-hydroxyethyl)-N′-acetic acid (HBMA); and N,N′-bis(5-deoxypyridoxyl)ethylenediamine-N,N′-diacetic acid (DPLED). The biodistribution of the radiometal complexes were carried out in rats and an imaging study was performed in a non-human primate. The rapid clearance of the lipophilic complexes from blood and through the hepatobiliary system was easily demonstrated; as well, the hydrophilic complexes were cleared rapidly through the urinary tract. Positron emission tomographic images were generated from a study in a primate after administration of ⁶⁸Ga-t-butyl HBED. These images well demonstrate the efficient liver accumulation and rapid hepatobiliary clearance (< 1 h) and well differentiate images of the liver and gall bladder.     

Characterization of DNA restriction and modification activities in Neisseria species

Type II restriction endonuclease activities detected in various Neisseria species were characterized for sequence specificity and precise site of cleavage. NsiCI isolated from N. sicca C351 cleaves the sequence 5′-GAT↓ATC-3′ (EcoRV isoschizomer); NmeCI from N. meningitidis C114 and NphI from N. pharyngis C245 cleave 5′-N↓GATCN-3′ (MboI isoschizomers); NgoPII and NgoPIII from N. gonorrhoeae P9-2 cleave at 5′-CC↓GCGG-3′ (SacII isoschizomer) and 5′-GG↓CC-3′ (HaeIII isoschizomer), respectively. Chromosomal DNA isolated from these strains and two other N. meningitidis strains (which lacked detectable endonuclease activities), was found to be refractive to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases.     

Potential radiopharmaceuticals for the diagnosis of biliary atresia and neonatal hepatitis: EHPG and HBED chelates of 67Ga and 111In

Radiopharmaceuticals suitable for use in cases where delayed excretion of hepatobiliary tracer can occur, were formulated from indium-111 and gallium-67 and the chelating agents N,N′-ethylene-bis-[o-hydroxyphenylglycine], (EHPG) and N,N′-bis-[2-hydroxybenzyl] ethylenediamine-N,N′-diacetic acid, (HBED). The hepatobiliary excretion of these ⁶⁷Ga and ¹¹¹In chelates was optimised by using chelators which had substituents in the phenolic ring; halogen substituents imparted the most favourable characteristics.     

Carbohydrate binding studies on the lectin from Datura stramonium seeds

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (M_r = 86,000) possesses two carbohydrate-binding sites for N,N′,N′',N?-tetraacetylchitotetritol/mol protein, with an apparent K_a = 8.7 × 10³M^?1 at 4 °C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N′- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N′,N?-triacetylchitotriose was over 6-fold more potent than the disaccharide (N′,N′-diacetylchitobiose) which, in turn, was 90 times more reactive than N-acetyl-d-glucosamine.N-Acetyllactosamine [β-d-Gal-(1 → 4)-d-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N′-diacetylchitobiose. The requirement for an N-acetyl-d-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.     

Ester hydrolyses catalyzed by modified cyclodextrins

Cyclohexaamylose-N-(N,N′-dimethylaminoethyl)acetohydroxamic acid and cyclohexaamylose-N-(4-imidazolemethyl)acetohydroxamic acid were synthesized. Their catalytic power is greater than that of cyclohexaamylose or of cyclohexaamylose-N-methylhydroxamic acid. In addition, optical selectivity was exhibited in the hydrolysis of d- and l-acetylphenylalanine p(m)-nitrophenyl esters by cyclohexaamylose-N-(N,N′-dimethylethyl)acetohydroxamic acid, and cycloheptaamylose.     

Nicotine N-oxides

Nicotine-1-N-oxide, trans and cis isomers of nicotine-1′-N-oxide and of nicotine-1,1′-di-N-oxide have been prepared and characterised by NMR, MS and reduction to nicotine. The trans and cis isomers of nicotine-1′-N-oxide have been identified in leaves, stems and roots of Nicotiana tabacum, N. affinis and N. sylvestris.     

Rate-limiting step in oxidation of physiological and artificial reductants by Azotobacter vinelandii membrane vesicles.

The respiration of Azotobacter vinelandii membrane vesicles was investigated in order to determine the partial rates of electron fluxes at each segment of its branched respiratory chain. It is concluded that under physiological conditions only 20 to 30% of the total flux is carried through the c₄, c₅ → a₁,o chain. Steady state analysis indicates that the limited capacity of the chain is due to the slow rate of oxidation of the cytochromes c by the a₁,o oxidases. This rate-limiting step is bypassed by the artificial electron donors, ascorbate-2,6-dichlorophenol indophenol and ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine, which directly reduce the highly active a₁,o oxidases. During the oxidation of ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine by the membrane vesicles, an accumulation of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine occurs. Such accumulation of positively charged molecules should lead to a generation of a membrane potential. This fact and previous data concerning coupling site III of A. vinelandii are discussed.     

Targeting radiopharmaceuticals—II. Evaluation of new trivalent metal complexes with different overall charges

Three new multidentate ligands designed to have high affinity for Ga³⁺, In³⁺ and Fe³⁺ have been synthesized, N-(2-hydroxybenzyl)-N′-pyridoxylethylenediamine-N,N′-diacetic acid (HBPLED), N-(2-hydroxy-3,5-dimethylbenzyl)-N′-(3-hydroxy-1,2,5-trimethyl-4-pyridylmethyl)ethylenediamine-N,N′-diacetic acid (Me₄HBPLED) and N,N′-bis(3-hydroxy-1,2,5-trimethyl-4-pyridylmethyl) ethylenediamine-N,N′-diacetic acid (DMPLED). These ligands give metal-ligand (M–L) complexes with (M = Ga³⁺, In³⁺, Fe³⁺) overall charges of −1, 0 and + 1, respectively. The ⁶⁷Ga, ⁶⁸Ga and ¹¹¹In complexes of each of the three ligands and the ⁵⁹Fe complex of Me₄HBPLED were prepared, characterized and their biodistributions determined in rats after intravenous injection. Despite the differences in overall charge, the biological behavior of all three ¹¹¹In complexes were similar, in that the radioactivity cleared rapidly via the kidney. The biodistributions of the ⁶⁸Ga and ⁶⁷Ga complexes were comparable to that of the ¹¹¹In complex counterpart. Also, the ⁵⁹Fe-Me₄HBPLED biodistribution was not significantly different from those of the ⁶⁸Gaand ¹¹¹In-Me₄HBPLED. The renal clearance rate seems insensitive to the overall M-L charge; suggesting that the hydrophilic periphery of the ligand rather than the overall molecular charge determines the biological fate (with respect to renal clearance).     

FT-IR and 1H NMR spectroscopic evidence of sugar ring conformational change in NH4GpG on complexation to form cis-[Pt(NH3)2(GpG)]+

The conformational change of the ribose ring in NH₄GpG and cis-[Pt(NH₃)₂(GpG)]⁺ was confirmed by FT-IR spectroscopic evidence as being C2′-endo, C3′-endo, anti, gg sugar ring pucker in the solid state. These results were compared with ¹H NMR spectral data in aqueous solution. The FT-IR spectrum of NH₄GpG shows marker bands at 802 cm^?1 and 797 cm^?1 which are assigned to the C3′-endo, anti, gg sugar-phosphate vibrations of ribose (?pG) and ribose (Gp?), respectively. The FT-IR spectrum of cis-[Pt(NH₃)₂(GpG)]⁺ (with N7N7 chelation in the GpG sequence) shows a marker band at 800 cm^?1 which is assigned to the C3′-endo, and a new shoulder band at 820 cm^?1 related to a C2′-endo ring pucker. The ribose conformation of (?pG) moiety in NH₄-GpG, C3′-endo, anti, gg changes into C2′-endo, anti, gg when a platinum atom is chelated to N7N7 in the GpG sequence.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.?coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.     

Nucleic acid-specific photoactivation of oligodeoxyribonucleotides labeled with deuterated dihydro-N,N,N′,N′-tetramethylrhodamine using green light

We developed a simple protocol for high-yielding synthesis of conjugates of a deuterated dihydro-N,N,N′,N′-tetramethylrhodamine (F*) with oligodeoxyribonucleotides and a 2′-OMe RNA (a representative nuclease-resistant, chemically modified oligonucleotide) using easily accessible starting materials including NaBD₄ and conjugates of oligonucleotides with N,N,N′,N′-tetramethylrhodamine (F). These compounds were found to be stable in air and insensitive to light at 525, 635 and 650 nm, whereas slow activation occurs upon their exposure to 470 nm light. However, at the conditions of the templated reaction, in the presence of a target nucleic acid and a photocatalyst based on the eosin structure, the F* is oxidized forming fluorescent F. This reaction is >30-fold faster than the background reaction in the absence of the template. Moreover, the presence of a single mismatch in the target nucleic acid slows down the templated reaction by eightfold. These activatable dyes can potentially find applications as nucleic acid-specific probes for super-resolution imaging in live cells.     

The DNA sequence on bacteriophage G4 recognized by the Escherichia coli B restriction enzyme.

Bacteriophage G4 possesses a single EcoB site located in the overlap between restriction fragments HinfI-12 and HaeIII-6. The sequence 5′-T-G-A … 8N … T-G-C-T occurs once in this segment and nowhere else in the DNA sequence of G4. Four independent G4 mutants that were not restricted by Escherichia coli B possessed the sequence 5′-T-G-A … 8N … T-G-C-C. The common sequence shared by the previously mapped EcoB sites on φXsB1, simian virus 40, f1, and fd DNAs is 5′-T-G-A … 8N … T-G-C-T … 9N … T. However, the sequence in the region of the G4 EcoB site contains an A instead of the final T conserved in these other examples. When the G4 EcoB site is aligned with the other EcoB sites, there are no conserved residues within 50 bases of the common sequence, 5′-T-G-A … 8N … T-G-C-T, except for those seven residues. The analysis of the EcoB site on G4 provides further evidence that only those seven bases are recognized by the E. coli B restriction enzyme.     

Radioimmunoassay of N'-nitrosonornicotine.

Antibodies specific for the precarcinogen N′-nitrosonornicotine (NNN) were obtained in rabbits immunized with a N′-nitroso-5′-carboxynornicotine-human serum albumin conjugate. The NNN derivative was synthesized by reduction and nitrosation of 2′,3′-dehydronornicotine and covalently conjugated to the macromolecule with the use of a carbodiimide. A radioimmunoassay was developed which could detect as little as 0.2 ng of NNN. Analyses of plasma supplemented with NNN indicated that it was possible to detect 2 ng NNN/ml sample. Nicotine, cotinine, pyridine, and pyrrolidine derivatives are ineffective inhibitors of the antigen-antibody reaction. N′-Nitrosoanabasine inhibits effectively, but this derivative has been reported not to occur in tobacco. The radioimmunoassay has been used to monitor nitrosation of nornicotine and anabasine under chemical conditions and to determine the rate constants for the reactions.     

The molecular structure and magnetic properties of the dimeric N,N′-ethylenebis(salicylamine)Fe(II)-μ-methoxo-N,N′-ethylene(o-hydroxylphenylglycine)salicylamine Fe(III): A complex with a μ-monodentate acetato bridge

We have prepared and characterized a dimeric complex of the formula Fe₂L(OCH₃)L′·solvent where L = N,N′-ethylenebis(salicylamine) and L′ = N,N′-ethylene(o-hydroxylphenylglycine)salicylamine. Crystals of the methanol solvate were found to be orthorhombic, space group P2nb, with a = 13.748 (5), b = 22.426(9), c = 11.762(6) Å and d_calc = 1.41 g/cm³. Least squares refinement of 966 reflections gave a final R factor of 0.066. The structure shows that the two iron atoms are coordinated to two different ligands and are bridged by a methoxide ion and a μ-monodentate acetate oxygen from the pendant arm of L′. The magnetic susceptibility was measured over the range 2–300 K and gave a spin coupling constant J = −8.3 cm⁻¹. The significance of acetate bridges to the exchange coupling pathway is discussed.     

Sth132I,a novel class-IIS restriction endonuclease of Streptococcus thermophilus ST132

The Sth132I restriction endonuclease (R.Sth132I) was detected in Streptococcus thermophilus ST132 and purified to near homogeneity by heparin Sepharose CL-6B affinity chromatography. Fragments from Sth132I digestion of plasmid DNA were subcloned into pUC19 in Escherichia coli DH5α and sequenced. Sequence analysis of inserts and their ligation junction sites revealed that Sth132I is a novel class-IIS restriction endonuclease, which recognizes the non-palindromic sequence5′-CCCG(N)₄-3′3′-GGGC(N)₈-5′.     

Inhibitory properties of N-nicotinyl-N' (substituted) urea analogs of NAD.

A series of N-nicotinyl- and N-isonicotinyl-N′-(substituted) ureas were synthesized by a condensation of the appropriate amide with various alkyl or aryl isocyanates, and examined as potential antimetabolites of nicotinamide. No significant microbial toxicity was observed; however, several derivatives in the nicotinyl series proved to be toxic to fish, especially N-nicotinyl-N′-hexylurea which possessed a TL₅₀ of 27 μg/ml. The toxicity was reversed by supplements of nicotinamide but not tryptophan, and may be due to the formation of an inactive NAD complex through the mediation of NADase. Evidence for this possibility is presented by the formation in vitro of an N-nicotinyl[7-¹⁴C]N′-ethylurea-NAD analog which was characterized through spectral, chemical, and chromatographic studies.     

Heterotrophic activity of bacterioplankton along the gradients of the chlorophyll <Emphasis Type="Italic">a</Emphasis> concentration and water temperature in marine and limnetic ecosystems

The dependence of the heterotrophic activity of bacterioplankton (V, μg C L^–1 h^–1) on the concentration of chlorophyll a (Chl, μg L^–1) and the water temperature (T) was examined for lakes (37°29′–80°36′ N) and marine polar waters (69°16′–80°36′ N). It was shown that ~76% of the V variations was related to changes in Chl and T.     

Phosphatidylinositol 3′-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3′-kinase (PI 3′-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3′-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3′-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3′-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca²⁺ levels that can be effected by Ca²⁺ mobilized from intracellular stores in the absence of Ca²⁺ influx regulate PA-induced chemotaxis. Furthermore, PI 3′-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP₃), suggesting that PI 3′-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3′-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3′-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Phenylpropanoid amides from Alisma orientalis and their protective effects against H2O2-induced damage in SH-SY5Y cells

Five new phenylpropanoid amides, including N-trans-feruloyl-N′-cis-feruloyl-cadaverine (1), N,N′-trans-diferuloyl-3-oxo-cadaverine (2), N-trans-feruloyl-N′-cis-feruloyl-3-hydroxy-cadaverine (3), N,N′-cis-diferuloyl-3-hydroxy-cadaverine (4), N-trans-p-coumaroyl-N′-trans-feruloyl-3-hydroxy-cadaverine (5), were isolated from Alisma orientalis together with four known analogues. Their structural elucidations were conducted by using 1D and 2D NMR and HRESIMS spectroscopic analyses. The isolated compounds were assayed for their inhibitory activities against HCE-2, anti-oxidant effects, and their protective effects on H₂O₂-induced damage in human dopaminergic neuroblastoma cells (SH-SY5Y). Compounds 3, 6, and 7 displayed moderate anti-oxidant activities with IC₅₀ values in the range of 36.9–40.7 μM. Compound 5 showed significant protective activity, while compounds 1, 2, 4, 7, and 8 showed moderate protective activities.     

5-Methyl coumarins and a new phenol from Nassauvia pyramidalis and N. digitata

The aerial parts of Nassauvia pyramidalis contain 5′epi-triptiliocoumarin and a new phenolic compound. N. digitata afforded the 5′-epi-triptiliocoumarin and triptiliocoumarin itself.